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A Radiation Hybrid Map of Mouse Chromosome 13
Genomics 57, 365–370 (1999) Article ID geno.1998.5205, available online at http://www.idealibrary.com on
A Radiation Hybrid Map of Mouse Chromosome 13 Rosemary W. Elliott,1 Kenneth F. Manly, and Colleen Hohman Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, New York State Department of Health, 666 Elm Street, Buffalo, New York 14263 Received September 15, 1997; accepted December 18, 1997
A mouse radiation hybrid (RH) panel was used to make a framework map for the entire length of mouse chromosome (Chr) 13. Forty-one loci were typed, and while most used primers flanking simple sequence repeats, some genes were included. The most proximal and distal loci are D13Mit132 and D13Mit35. The estimate of map length for Chr 13 is 1328 cR. The map is compared with the same set of loci from the consensus map for Chr 13, which is 70 cM in length, and also with a recombinational map derived from an intraspecies cross typed for many of the same loci. The mouse RH panel gave good resolution for Chr 13 and at the distal end allowed separation of previously nonrecombinant markers that are present on a single 620-kb YAC clone. Data analysis was performed using the RH option for Map Manager QT. This framework RH map of Chr 13 is the second of a series of RH maps for mouse chromosomes. q 1999 Academic Press
INTRODUCTION
The use of radiation hybrids (RHs) to generate longrange maps of chromosomes was an important step in human genetics. The map of human chromosome 21 was the first example (Cox et al., 1990). This study also included a statistical approach to measuring the proportion of breaks, u, between two loci, based on the number of hybrids containing DNA breaks between the loci. This statistic is modified by a mapping function that converts the distance to centiRays (cR). Finally, the study provided a lod score function representing the degree of support for linkage between two loci. Since the initial studies, radiation maps have been produced for other single human chromosomes, such as a map of human chromosome 11 with over 500 loci (James et al., 1994) and human chromosomes X, 18, 12p, and 9p (Bouzyk et al., 1996; Giacalone et al., 1996; Kumlien et al., 1996; Raeymaekers et al., 1995). The GeneBridge Panel 4 (Walter et al., 1994) has been used 1 To whom correspondence should be addressed. Telephone: (716) 845-3277. Fax: (716) 845-8169. E-mail: [email protected]. buffalo.edu.
to generate a map that is used by many workers (Hudson et al., 1995). A framework map of the whole human genome has also been constructed using this panel (Gyapay et al., 1996). An STS map of the human genome was generated using the Stanford G3 panel of 83 hybrids (Stewart et al., 1997). A RH panel recently developed by Dr. Peter Goodfellow (Schmitt et al., 1996) is now available for the mouse. Mouse cells were exposed to 3000 rads of X irradiation and fused with the nonirradiated hamster cell line A23. DNA was isolated from 100 cell hybrids. Recombinational maps are available for the mouse genome, and several interspecies maps now include many loci (Rowe et al., 1994; Kozak et al., 1990; Copeland et al., 1993). However, there are still many loci that, due to lack of genetic variation, cannot be typed on genetic maps. Furthermore, map order for markers typed on different crosses is tentative. These problems can be remedied using radiation hybrids because all loci that have been sequenced can be typed on RH panels, as there is no requirement for genetic variation. To begin to use RH maps to answer genetic questions, it is necessary to generate framework maps for all chromosomes. In this study we present a framework map of mouse chromosome (Chr) 13, using the Goodfellow hybrid panel (McCarthy et al., 1997). We also describe a new computer program based on Map Manager (Manly, 1993) that will hold the typing data, generate map distances in centirays, calculate lod scores (Cox et al., 1990), and allow linkage of new markers to be tested and established. MATERIALS AND METHODS Radiation hybrids. A panel of DNA from 100 mouse–hamster RH clones was purchased from Research Genetics, Inc. DNA samples were arrayed in 96-well Falcon microtest plates. For each amplification, 20 ng of DNA from each clone was transferred, using a multichannel pipette, to a second plate. PCR primers for loci defined by amplification of markers described by MIT (Dietrich et al., 1994) were obtained from Research Genetics. We thank Dr. Richard Swank of the Roswell Park Cancer Institute (RPCI) for primers for Cf2r (coagulation factor 2 receptor) and Dhfr (dihydrofolate reductase). The primer pair for Mekk (MEK kinase) was found by searching the Mouse Genome Database and was synthesized at the RPCI Biopolymer Facility.
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0888-7543/99 $30.00 Copyright q 1999 by Academic Press All rights of reproduction in any form reserved.
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PCR. PCR using the above primers was carried out in multiwell plates in the PTC-100-96 thermal cycler (M. J. Research, Inc.) in a 10-ml volume containing 20 ng of genomic DNA, 1 to 2.5 pmol of each primer and 0.08 unit of Taq polymerase. The mixture also contained 0.05% bovine serum albumin, 1.5 mM MgCl2 , 10 mM Tris–HCl, 50 mM KCl, and 1 ml 2% cresol red dye in 60% sucrose. The cycling parameters used were 947C for 3 min, followed by 947C for 45 s, 557C for 35 s, 727C for 30 s for 38 cycles, with the last extension time 5 min. Analysis of the reaction product was performed by electrophoresis on 2% Seakem LE agarose gels (FMC Products) in 0.51 Tris-borate buffer, pH 8.3. The apparatus was purchased from Owl Scientific. The wells were formed using three 42-slot multiwell combs, and 3 ml of each reaction mixture was loaded using a multichannel pipette. Electrophoresis was carried out for 45–60 min at 200 V and fragments were visualized after ethidium bromide staining. Gels were photographed on thermal paper K65HM (Mitsubishi) using the Alpha Imager 2000 Digital Imaging System (Alpha Innotech Corp.). Retyping of questionable data was performed using AmpliTaq Gold polymerase as suggested by the manufacturer. Use of this product caused a decreased signal for misprimed hamster fragments and allowed us to type samples with greater confidence. Data analysis. Genetic data were stored in an enhanced version of Map Manager QTb15, available at http://mcbio.med.buffalo.edu/ mmQT.html, which is itself an enhanced version of Map Manager Classic (Manly, 1993). The enhanced version contains a radiation hybrid analysis option. The typings were entered in a data window similar to the data window used to analyze genetic backcrosses. Map distances and lod scores were calculated according to Cox et al., (1990). The program can store data, generate a map with distances in centirays, present a statistical analysis of data for each locus, store comments, and generate haplotype figures. The distance and lod score are based on two-point analysis from adjacent markers. The ability to adjust the position of the markers expands the capacity for analysis. Genetic analyses were performed using this program, which was also used to draw the RH map shown in Fig. 2. Typing and retention data are available at http://www.jax.org/resources/doc uments/cmdata/rhmap/rh.html.
RESULTS
Choice of PCR primers. To develop the framework map, loci defining SSRs (Dietrich et al., 1994) were chosen to span the full length of Chr 13, including the most proximal and most distal loci. Preference was given to SSRs that had already been typed together in the backcross (ICR/Ha 1 B57BL/6Ha) 1 C57BL/6Ha (IBB) and to those that identify genes. Typing. DNA samples from the panel were amplified using each set of primers, and the presence or absence of specific amplification products was determined after agarose gel electrophoresis of the amplified product. Data were entered into Map Manager QT and analyzed using the RH option. The typings for 41 loci are shown in Fig. 1, which indicates which loci were amplified in each of the 100 hybrids. The assumption is made that if adjacent loci are retained in the same hybrid, then the intervening DNA is also retained. Under this assumption, Fig. 1 illustrates the lengths of DNA fragments from Chr 13 retained within each RH. The lengths range from the full length of Chr 13 in RH lines 56 and 79 to a single locus in nine RH lines. Eleven hybrids appear to contain no DNA from mouse Chr 13, and 1 hybrid contains eight fragments. However, as more loci are typed, short stretches of DNA from Chr 13 may be identified in some of these hybrids. The
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range and pattern of lengths of fragments are similar to those found in a RH panel for human chromosome X (Kumlien et al., 1996). For instance, there are several cases in which both a long fragment and a short adjacent fragment are present in the same hybrid, but the intervening DNA appears to be absent. Data from such hybrids were checked for errors both by rereading the pictures and by retyping the samples. The map. The RH map for the loci used in Fig. 1 is the central map in Fig. 2. The order of most loci was previously known. Typing data for each locus were therefore placed in the predicted position in the data window. Locus order was tested by moving the data for a locus to a more proximal position and noting the change in the number of apparent breaks and in the lod score for linkage for markers affected by the change. The test was continued by moving the locus to a more distal position and noting the changes in the same parameters. The final position was chosen to minimize the number of breaks and maximize the LOD for linkage. For all of the 41 markers, the positions are based on odds for linkage greater than 10,000:1. The RH map was compared to a map of many of the same loci derived from the intraspecies backcross (ICR/ Ha 1 C57BL/6Ha) 1 C57BL/6Ha [(IB)B] (Jacoby et al., 1994) on the right of Fig. 2. This map uses data only from female F1 hybrids. The loci present in both maps have the same order, except for the adjacent markers D13Mit1 and D13Mit216. A further comparison of the RH map was made with the same loci from the most recent consensus map (Justice and Stephenson, 1996). This consensus map was constructed using all published data for Chr 13. An initial map, based mainly on multilocus backcrosses and the MIT F2 cross (Dietrich et al., 1994), was generated, and data from smaller crosses and RI strains were then incorporated using interpolation. The order of markers from different crosses is not as reliable as is the order of markers from the same cross. On the left of Fig. 2 is a map of the consensus positions of the loci typed on the RH map and there is generally good agreement between the two maps. However, the locus order in the consensus map differs from the locus order in the RH map at the proximal end of Chr 13. The RH map shows that the most proximal locus is D13Mit132, and the backcross map is consistent with this, but this locus is placed more distally in the consensus map. The order of several of the other proximal loci also differs from that found in the consensus map and in the map obtained from the MIT F2. In a more distal region, the consensus map shows that Cf2r is proximal to Dhfr. In the RH map, Dhfr is proximal to Cf2r, consistent with recent recombinational data for this region (Seymour et al., 1996). Fragment retention. The retention of loci is determined by the fraction of hybrid cells containing a given locus. Retention for Chr 13 loci in this RH panel is shown in Fig. 3 and ranges from 19 to 44%, except for the most proximal marker, which is retained by 61%
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MOUSE CHR 13 RADIATION HYBRID MAP
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FIG. 1. Cell lines showing presence of DNA for Chr 13 loci. Cell lines are numbered as received from the vendor and as listed in Map Manager QT files. Loci are listed in map order, with the centromere at the top. A vertical line indicates the presence of specific mouse DNA, as demonstrated by its ability to be amplified with primers associated with the listed loci. Continuous lines indicate that there is no evidence of a break in DNA present in that line.
of the lines. This is consistent with high retention at centromeric loci found for human chromosomes. The average retention for 41 loci is 29.5%. There are slight variations along the chromosome, but no dramatic changes. Retention for the distal loci ranges from 19 to 32%, while retention for the proximal half of the chromosome appears higher, 25 to 40%, if the most proximal locus is ignored. These values do not appear to differ from retention estimates observed in human hybrid panels. DISCUSSION
A radiation hybrid framework map of mouse Chr 13 was generated. The map was obtained using a new program to analyze RH data, the RH option for Map Manager QT. In this initial use of the program the probable order of the markers was known. The order predicted from the data was tested first by minimizing the number of breaks and second by maximizing the lod scores for linkage between the loci. The map obtained using these criteria was compared to the consensus map. The locus order was the same
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except for the proximal 10 map units in the consensus map and for two genes in the distal portion of Chr 13. The order of loci defined by SSRs in the consensus map is based largely on the map of the MIT F2 cross (Dietrich et al., 1994). A search of their database showed that several of the proximal markers were typed as dominant markers, when one homozygote could be typed unequivocally, but the heterozygotes could not be distinguished from the other homozygote. This means that these markers were placed on the map with very low odds. The RH map places these proximal loci with odds greater than 100,000 to 1. Furthermore, the order for many of the loci is confirmed by the (IB)B backcross. Further confirmation comes from physical maps of the region generated during the analysis of the beige mutation, now Lyst (Misumi et al., 1997; Perou et al., 1997). One of the crosses (Misumi et al., 1997) places D13Mit216 proximal to D13Mit1, in agreement with the RH map, while the other places D13Mit1 proximal, in agreement with our results for the genetic cross. Reversing the order of these two markers in the RH map generates four more apparent chromosomal breaks. A difference between the RH map and the recombina-
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FIG. 2. Maps of Chr 13. All maps are oriented with the centromere at the top. The center map was derived from the data presented in Fig. 1, using calculations as described (Cox et al., 1990). Positions of loci are given in centirays. The map on the left was obtained from the consensus map, using the distance from the centromere in centimorgans as the map position for each locus. The horizontal bars on the vertical line of the map represent 10-cM intervals. To illustrate order differences, proximal loci in these two maps are linked by dashed lines. On the right is a recombinational map for Chr 13, obtained from intraspecies cross (IB)B. Most loci in this cross were also typed on the RH DNA. Map distances represent percentage of recombination obtained after assigning a position of 0 to the most proximal marker.
tional maps is that the relative distances for some intervals are altered. The fraction of the map length occupied by the proximal 15 loci, to D13Mit116, is much greater for the RH map (41%) than either in the consensus map or for the same interval in the map generated using the (IB)B cross (16.6%). Eliminating the proximal locus, which has a high retention frequency, and using the interval D13Mit216 to D13Mit116 gives 33.9 and 11.6% of the RH and recombinational maps, respectively. The basis for this difference is not clear, but a
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number of factors could contribute, some from the data for the RH map and some from the recombinational data. If X-ray-induced chromosomal breaks occur randomly, then the RH map should represent a physical map. However, if breaks are not random in some regions, this could affect relative distances. For Chr 13, the retention frequency appears slightly higher in the proximal regions (Fig. 3) and this may be a contributing factor. Errors in the typing of some loci could lengthen
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MOUSE CHR 13 RADIATION HYBRID MAP
FIG. 3. Retention frequency plot. The number of lines amplifying each locus was obtained from Map Manager QT and is plotted against the loci, which are presented in the same order as in Figs. 1 and 2. Each bar in the histogram is to the right of a number, which represents the order of loci in Figs. 1 and 2.
the RH map by introducing spurious chromosomal breaks. To remove typing errors, we have retyped several loci, particularly those that indicate the retention of short DNA fragments amplified by only a single locus, such as D13Mit132. On the other hand, with only 41 loci typed, not all the bins have been defined, so the map length could increase as more loci are added and more short pieces of DNA are identified, each with two breaks that would contribute additional map length. We have previously reported compression of the distal end of the recombinational map of Chr 13 in the (IB)B cross (Yen et al., 1997). This compression can be seen at the distal end of the recombinational map in Fig. 2. There is no recombination between D13Mit148 and the loci distal to it, while the RH map shows that the region occupies 181 cR. There is also map compression on distal Chr 13 in crosses involving Mus spretus, possibly due to the presence of a ribosomal gene cluster, Rnr13 (Eicher and Shown, 1993). We have chosen to use the (IB)B cross in Fig. 2 because, unlike crosses involving M. spretus, there appeared to be no map compression for proximal Chr 13 loci. Map compression can be seen in the map obtained from (C57BL/6J 1 SPRET/ Ei) 1 C57BL/6J (http://www.jax.org/resources/documents/cmdata/maps/MapList.html), in which D13Mit1 and D13Mit3 are nonrecombinant, while they would be about 8 map units apart in the (IB)B backcross map and are 331 cR apart on the RH map. Map compression on proximal Chr 13 has also been observed in a cross involving male meiosis, C57BL/6Ha 1 (ICR/Ha 1 C57BL/6Ha), for the two strains used in the (IB)B cross (Elliott et al., 1997). Despite the recombination observed at proximal Chr 13 in the (IB)B cross, it is possible that some recombinational compression does exist in this region, as suggested by the differences in fractional map length described above. The length estimate of the map is 1328 cR. Map lengths are dependent on the amount of radiation used to generate the hybrids. It is thus difficult to compare map lengths between different hybrid sets. As some of the map intervals in Fig. 2 are quite long, it appears likely that other breaks associated with short chromosomal fragments or short deletions in long fragments may be present in some of these intervals. Finding such
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breaks would increase the estimate of chromosomal length. Map length estimates are also affected by errors, which vary in their effects depending on whether the error is a false positive or a false negative. Errors that generate new breaks increase the apparent map length, while errors that eliminate breaks decrease the apparent map length. In this study, all results that were not internally consistent were retested and independently assessed by two individuals. The mouse genome contains approximately 3000 Mb of DNA, so an average-sized chromosome like Chr 13 should contain about 150 Mb. In making the RH map for Chr 13, we have identified 445 radiation-induced breaks in the 100 cell lines, which is a minimum estimate. On this map, 1 cR represents 113 kb, and the average resolution of the map is 3.8 Mb per interval. No report of genetic recombination between the most distal SSR markers, D13Mit77 and D13Mit35, has yet been made. These markers are on the same 620-kb YAC clone y60E7 (Yen et al., 1997), but are separated in the RH panel by breaks in six hybrids. The RH map thus allows these two loci to be ordered relative to neighboring loci and orients the map of YAC y60E7 to the chromosome. Two loci on the map, D13Rp3 and D13Rp4, are about 4 kb apart (Yen et al., 1997). There is one break between them, which does not order them relative to the rest of the chromosome. These loci, as well as D13Mit196, are on YAC y72G2. D13Mit53 maps between these markers, but does not amplify YAC y72G2, suggesting that this YAC carries a deletion between D13Rp4 and D13Mit196. Three of the loci, D13Mit161, 169, and Cf2r, have been used to obtain a physical map around pearl ( pe) (R. Swank, unpublished). The order obtained from the RH map is the same as that obtained from the physical map. Similarly, several of the loci have been used to obtain a physical map of the region around beige (bg, now Lyst) (Barbosa et al., 1996, Misumi et al., 1997, Perou et al., 1997). The order for the physically mapped loci is also the same as that found here for the RH panel. ACKNOWLEDGMENTS This work was supported by NIH Grant GM33160 to R.W.E., also by NCI Core Grant CA16056-21 to the RPCI. The development of Map Manager was partly supported by NIH Grant HG00330 to The Jackson Laboratory.
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A Radiation Hybrid Map of Mouse Chromosome 13 Rosemary W. Elliott,1 Kenneth F. Manly, and Colleen Hohman Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, New York State Department of Health, 666 Elm Street, Buffalo, New York 14263 Received September 15, 1997; accepted December 18, 1997
A mouse radiation hybrid (RH) panel was used to make a framework map for the entire length of mouse chromosome (Chr) 13. Forty-one loci were typed, and while most used primers flanking simple sequence repeats, some genes were included. The most proximal and distal loci are D13Mit132 and D13Mit35. The estimate of map length for Chr 13 is 1328 cR. The map is compared with the same set of loci from the consensus map for Chr 13, which is 70 cM in length, and also with a recombinational map derived from an intraspecies cross typed for many of the same loci. The mouse RH panel gave good resolution for Chr 13 and at the distal end allowed separation of previously nonrecombinant markers that are present on a single 620-kb YAC clone. Data analysis was performed using the RH option for Map Manager QT. This framework RH map of Chr 13 is the second of a series of RH maps for mouse chromosomes. q 1999 Academic Press
INTRODUCTION
The use of radiation hybrids (RHs) to generate longrange maps of chromosomes was an important step in human genetics. The map of human chromosome 21 was the first example (Cox et al., 1990). This study also included a statistical approach to measuring the proportion of breaks, u, between two loci, based on the number of hybrids containing DNA breaks between the loci. This statistic is modified by a mapping function that converts the distance to centiRays (cR). Finally, the study provided a lod score function representing the degree of support for linkage between two loci. Since the initial studies, radiation maps have been produced for other single human chromosomes, such as a map of human chromosome 11 with over 500 loci (James et al., 1994) and human chromosomes X, 18, 12p, and 9p (Bouzyk et al., 1996; Giacalone et al., 1996; Kumlien et al., 1996; Raeymaekers et al., 1995). The GeneBridge Panel 4 (Walter et al., 1994) has been used 1 To whom correspondence should be addressed. Telephone: (716) 845-3277. Fax: (716) 845-8169. E-mail: [email protected]. buffalo.edu.
to generate a map that is used by many workers (Hudson et al., 1995). A framework map of the whole human genome has also been constructed using this panel (Gyapay et al., 1996). An STS map of the human genome was generated using the Stanford G3 panel of 83 hybrids (Stewart et al., 1997). A RH panel recently developed by Dr. Peter Goodfellow (Schmitt et al., 1996) is now available for the mouse. Mouse cells were exposed to 3000 rads of X irradiation and fused with the nonirradiated hamster cell line A23. DNA was isolated from 100 cell hybrids. Recombinational maps are available for the mouse genome, and several interspecies maps now include many loci (Rowe et al., 1994; Kozak et al., 1990; Copeland et al., 1993). However, there are still many loci that, due to lack of genetic variation, cannot be typed on genetic maps. Furthermore, map order for markers typed on different crosses is tentative. These problems can be remedied using radiation hybrids because all loci that have been sequenced can be typed on RH panels, as there is no requirement for genetic variation. To begin to use RH maps to answer genetic questions, it is necessary to generate framework maps for all chromosomes. In this study we present a framework map of mouse chromosome (Chr) 13, using the Goodfellow hybrid panel (McCarthy et al., 1997). We also describe a new computer program based on Map Manager (Manly, 1993) that will hold the typing data, generate map distances in centirays, calculate lod scores (Cox et al., 1990), and allow linkage of new markers to be tested and established. MATERIALS AND METHODS Radiation hybrids. A panel of DNA from 100 mouse–hamster RH clones was purchased from Research Genetics, Inc. DNA samples were arrayed in 96-well Falcon microtest plates. For each amplification, 20 ng of DNA from each clone was transferred, using a multichannel pipette, to a second plate. PCR primers for loci defined by amplification of markers described by MIT (Dietrich et al., 1994) were obtained from Research Genetics. We thank Dr. Richard Swank of the Roswell Park Cancer Institute (RPCI) for primers for Cf2r (coagulation factor 2 receptor) and Dhfr (dihydrofolate reductase). The primer pair for Mekk (MEK kinase) was found by searching the Mouse Genome Database and was synthesized at the RPCI Biopolymer Facility.
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PCR. PCR using the above primers was carried out in multiwell plates in the PTC-100-96 thermal cycler (M. J. Research, Inc.) in a 10-ml volume containing 20 ng of genomic DNA, 1 to 2.5 pmol of each primer and 0.08 unit of Taq polymerase. The mixture also contained 0.05% bovine serum albumin, 1.5 mM MgCl2 , 10 mM Tris–HCl, 50 mM KCl, and 1 ml 2% cresol red dye in 60% sucrose. The cycling parameters used were 947C for 3 min, followed by 947C for 45 s, 557C for 35 s, 727C for 30 s for 38 cycles, with the last extension time 5 min. Analysis of the reaction product was performed by electrophoresis on 2% Seakem LE agarose gels (FMC Products) in 0.51 Tris-borate buffer, pH 8.3. The apparatus was purchased from Owl Scientific. The wells were formed using three 42-slot multiwell combs, and 3 ml of each reaction mixture was loaded using a multichannel pipette. Electrophoresis was carried out for 45–60 min at 200 V and fragments were visualized after ethidium bromide staining. Gels were photographed on thermal paper K65HM (Mitsubishi) using the Alpha Imager 2000 Digital Imaging System (Alpha Innotech Corp.). Retyping of questionable data was performed using AmpliTaq Gold polymerase as suggested by the manufacturer. Use of this product caused a decreased signal for misprimed hamster fragments and allowed us to type samples with greater confidence. Data analysis. Genetic data were stored in an enhanced version of Map Manager QTb15, available at http://mcbio.med.buffalo.edu/ mmQT.html, which is itself an enhanced version of Map Manager Classic (Manly, 1993). The enhanced version contains a radiation hybrid analysis option. The typings were entered in a data window similar to the data window used to analyze genetic backcrosses. Map distances and lod scores were calculated according to Cox et al., (1990). The program can store data, generate a map with distances in centirays, present a statistical analysis of data for each locus, store comments, and generate haplotype figures. The distance and lod score are based on two-point analysis from adjacent markers. The ability to adjust the position of the markers expands the capacity for analysis. Genetic analyses were performed using this program, which was also used to draw the RH map shown in Fig. 2. Typing and retention data are available at http://www.jax.org/resources/doc uments/cmdata/rhmap/rh.html.
RESULTS
Choice of PCR primers. To develop the framework map, loci defining SSRs (Dietrich et al., 1994) were chosen to span the full length of Chr 13, including the most proximal and most distal loci. Preference was given to SSRs that had already been typed together in the backcross (ICR/Ha 1 B57BL/6Ha) 1 C57BL/6Ha (IBB) and to those that identify genes. Typing. DNA samples from the panel were amplified using each set of primers, and the presence or absence of specific amplification products was determined after agarose gel electrophoresis of the amplified product. Data were entered into Map Manager QT and analyzed using the RH option. The typings for 41 loci are shown in Fig. 1, which indicates which loci were amplified in each of the 100 hybrids. The assumption is made that if adjacent loci are retained in the same hybrid, then the intervening DNA is also retained. Under this assumption, Fig. 1 illustrates the lengths of DNA fragments from Chr 13 retained within each RH. The lengths range from the full length of Chr 13 in RH lines 56 and 79 to a single locus in nine RH lines. Eleven hybrids appear to contain no DNA from mouse Chr 13, and 1 hybrid contains eight fragments. However, as more loci are typed, short stretches of DNA from Chr 13 may be identified in some of these hybrids. The
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range and pattern of lengths of fragments are similar to those found in a RH panel for human chromosome X (Kumlien et al., 1996). For instance, there are several cases in which both a long fragment and a short adjacent fragment are present in the same hybrid, but the intervening DNA appears to be absent. Data from such hybrids were checked for errors both by rereading the pictures and by retyping the samples. The map. The RH map for the loci used in Fig. 1 is the central map in Fig. 2. The order of most loci was previously known. Typing data for each locus were therefore placed in the predicted position in the data window. Locus order was tested by moving the data for a locus to a more proximal position and noting the change in the number of apparent breaks and in the lod score for linkage for markers affected by the change. The test was continued by moving the locus to a more distal position and noting the changes in the same parameters. The final position was chosen to minimize the number of breaks and maximize the LOD for linkage. For all of the 41 markers, the positions are based on odds for linkage greater than 10,000:1. The RH map was compared to a map of many of the same loci derived from the intraspecies backcross (ICR/ Ha 1 C57BL/6Ha) 1 C57BL/6Ha [(IB)B] (Jacoby et al., 1994) on the right of Fig. 2. This map uses data only from female F1 hybrids. The loci present in both maps have the same order, except for the adjacent markers D13Mit1 and D13Mit216. A further comparison of the RH map was made with the same loci from the most recent consensus map (Justice and Stephenson, 1996). This consensus map was constructed using all published data for Chr 13. An initial map, based mainly on multilocus backcrosses and the MIT F2 cross (Dietrich et al., 1994), was generated, and data from smaller crosses and RI strains were then incorporated using interpolation. The order of markers from different crosses is not as reliable as is the order of markers from the same cross. On the left of Fig. 2 is a map of the consensus positions of the loci typed on the RH map and there is generally good agreement between the two maps. However, the locus order in the consensus map differs from the locus order in the RH map at the proximal end of Chr 13. The RH map shows that the most proximal locus is D13Mit132, and the backcross map is consistent with this, but this locus is placed more distally in the consensus map. The order of several of the other proximal loci also differs from that found in the consensus map and in the map obtained from the MIT F2. In a more distal region, the consensus map shows that Cf2r is proximal to Dhfr. In the RH map, Dhfr is proximal to Cf2r, consistent with recent recombinational data for this region (Seymour et al., 1996). Fragment retention. The retention of loci is determined by the fraction of hybrid cells containing a given locus. Retention for Chr 13 loci in this RH panel is shown in Fig. 3 and ranges from 19 to 44%, except for the most proximal marker, which is retained by 61%
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FIG. 1. Cell lines showing presence of DNA for Chr 13 loci. Cell lines are numbered as received from the vendor and as listed in Map Manager QT files. Loci are listed in map order, with the centromere at the top. A vertical line indicates the presence of specific mouse DNA, as demonstrated by its ability to be amplified with primers associated with the listed loci. Continuous lines indicate that there is no evidence of a break in DNA present in that line.
of the lines. This is consistent with high retention at centromeric loci found for human chromosomes. The average retention for 41 loci is 29.5%. There are slight variations along the chromosome, but no dramatic changes. Retention for the distal loci ranges from 19 to 32%, while retention for the proximal half of the chromosome appears higher, 25 to 40%, if the most proximal locus is ignored. These values do not appear to differ from retention estimates observed in human hybrid panels. DISCUSSION
A radiation hybrid framework map of mouse Chr 13 was generated. The map was obtained using a new program to analyze RH data, the RH option for Map Manager QT. In this initial use of the program the probable order of the markers was known. The order predicted from the data was tested first by minimizing the number of breaks and second by maximizing the lod scores for linkage between the loci. The map obtained using these criteria was compared to the consensus map. The locus order was the same
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except for the proximal 10 map units in the consensus map and for two genes in the distal portion of Chr 13. The order of loci defined by SSRs in the consensus map is based largely on the map of the MIT F2 cross (Dietrich et al., 1994). A search of their database showed that several of the proximal markers were typed as dominant markers, when one homozygote could be typed unequivocally, but the heterozygotes could not be distinguished from the other homozygote. This means that these markers were placed on the map with very low odds. The RH map places these proximal loci with odds greater than 100,000 to 1. Furthermore, the order for many of the loci is confirmed by the (IB)B backcross. Further confirmation comes from physical maps of the region generated during the analysis of the beige mutation, now Lyst (Misumi et al., 1997; Perou et al., 1997). One of the crosses (Misumi et al., 1997) places D13Mit216 proximal to D13Mit1, in agreement with the RH map, while the other places D13Mit1 proximal, in agreement with our results for the genetic cross. Reversing the order of these two markers in the RH map generates four more apparent chromosomal breaks. A difference between the RH map and the recombina-
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FIG. 2. Maps of Chr 13. All maps are oriented with the centromere at the top. The center map was derived from the data presented in Fig. 1, using calculations as described (Cox et al., 1990). Positions of loci are given in centirays. The map on the left was obtained from the consensus map, using the distance from the centromere in centimorgans as the map position for each locus. The horizontal bars on the vertical line of the map represent 10-cM intervals. To illustrate order differences, proximal loci in these two maps are linked by dashed lines. On the right is a recombinational map for Chr 13, obtained from intraspecies cross (IB)B. Most loci in this cross were also typed on the RH DNA. Map distances represent percentage of recombination obtained after assigning a position of 0 to the most proximal marker.
tional maps is that the relative distances for some intervals are altered. The fraction of the map length occupied by the proximal 15 loci, to D13Mit116, is much greater for the RH map (41%) than either in the consensus map or for the same interval in the map generated using the (IB)B cross (16.6%). Eliminating the proximal locus, which has a high retention frequency, and using the interval D13Mit216 to D13Mit116 gives 33.9 and 11.6% of the RH and recombinational maps, respectively. The basis for this difference is not clear, but a
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number of factors could contribute, some from the data for the RH map and some from the recombinational data. If X-ray-induced chromosomal breaks occur randomly, then the RH map should represent a physical map. However, if breaks are not random in some regions, this could affect relative distances. For Chr 13, the retention frequency appears slightly higher in the proximal regions (Fig. 3) and this may be a contributing factor. Errors in the typing of some loci could lengthen
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FIG. 3. Retention frequency plot. The number of lines amplifying each locus was obtained from Map Manager QT and is plotted against the loci, which are presented in the same order as in Figs. 1 and 2. Each bar in the histogram is to the right of a number, which represents the order of loci in Figs. 1 and 2.
the RH map by introducing spurious chromosomal breaks. To remove typing errors, we have retyped several loci, particularly those that indicate the retention of short DNA fragments amplified by only a single locus, such as D13Mit132. On the other hand, with only 41 loci typed, not all the bins have been defined, so the map length could increase as more loci are added and more short pieces of DNA are identified, each with two breaks that would contribute additional map length. We have previously reported compression of the distal end of the recombinational map of Chr 13 in the (IB)B cross (Yen et al., 1997). This compression can be seen at the distal end of the recombinational map in Fig. 2. There is no recombination between D13Mit148 and the loci distal to it, while the RH map shows that the region occupies 181 cR. There is also map compression on distal Chr 13 in crosses involving Mus spretus, possibly due to the presence of a ribosomal gene cluster, Rnr13 (Eicher and Shown, 1993). We have chosen to use the (IB)B cross in Fig. 2 because, unlike crosses involving M. spretus, there appeared to be no map compression for proximal Chr 13 loci. Map compression can be seen in the map obtained from (C57BL/6J 1 SPRET/ Ei) 1 C57BL/6J (http://www.jax.org/resources/documents/cmdata/maps/MapList.html), in which D13Mit1 and D13Mit3 are nonrecombinant, while they would be about 8 map units apart in the (IB)B backcross map and are 331 cR apart on the RH map. Map compression on proximal Chr 13 has also been observed in a cross involving male meiosis, C57BL/6Ha 1 (ICR/Ha 1 C57BL/6Ha), for the two strains used in the (IB)B cross (Elliott et al., 1997). Despite the recombination observed at proximal Chr 13 in the (IB)B cross, it is possible that some recombinational compression does exist in this region, as suggested by the differences in fractional map length described above. The length estimate of the map is 1328 cR. Map lengths are dependent on the amount of radiation used to generate the hybrids. It is thus difficult to compare map lengths between different hybrid sets. As some of the map intervals in Fig. 2 are quite long, it appears likely that other breaks associated with short chromosomal fragments or short deletions in long fragments may be present in some of these intervals. Finding such
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breaks would increase the estimate of chromosomal length. Map length estimates are also affected by errors, which vary in their effects depending on whether the error is a false positive or a false negative. Errors that generate new breaks increase the apparent map length, while errors that eliminate breaks decrease the apparent map length. In this study, all results that were not internally consistent were retested and independently assessed by two individuals. The mouse genome contains approximately 3000 Mb of DNA, so an average-sized chromosome like Chr 13 should contain about 150 Mb. In making the RH map for Chr 13, we have identified 445 radiation-induced breaks in the 100 cell lines, which is a minimum estimate. On this map, 1 cR represents 113 kb, and the average resolution of the map is 3.8 Mb per interval. No report of genetic recombination between the most distal SSR markers, D13Mit77 and D13Mit35, has yet been made. These markers are on the same 620-kb YAC clone y60E7 (Yen et al., 1997), but are separated in the RH panel by breaks in six hybrids. The RH map thus allows these two loci to be ordered relative to neighboring loci and orients the map of YAC y60E7 to the chromosome. Two loci on the map, D13Rp3 and D13Rp4, are about 4 kb apart (Yen et al., 1997). There is one break between them, which does not order them relative to the rest of the chromosome. These loci, as well as D13Mit196, are on YAC y72G2. D13Mit53 maps between these markers, but does not amplify YAC y72G2, suggesting that this YAC carries a deletion between D13Rp4 and D13Mit196. Three of the loci, D13Mit161, 169, and Cf2r, have been used to obtain a physical map around pearl ( pe) (R. Swank, unpublished). The order obtained from the RH map is the same as that obtained from the physical map. Similarly, several of the loci have been used to obtain a physical map of the region around beige (bg, now Lyst) (Barbosa et al., 1996, Misumi et al., 1997, Perou et al., 1997). The order for the physically mapped loci is also the same as that found here for the RH panel. ACKNOWLEDGMENTS This work was supported by NIH Grant GM33160 to R.W.E., also by NCI Core Grant CA16056-21 to the RPCI. The development of Map Manager was partly supported by NIH Grant HG00330 to The Jackson Laboratory.
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