J. Schläpfer

A whole-genome radiation hybrid panel for bovine gene mapping

Although the idea of irradiation and fusion gene transfer was published more than 20 years ago (Goss and Harris 1975) and employed in an isolated mapping experiment ten years later (Willard et al. 1985), it was not systematically employed as a human gene mapping tool until resurrected by Cox and associates (1990) for constructing a high-resolution map of human Chr 21. Wholegenome radiation hybrid (WG-RH) mapping utilizing irradiated diploid human cells rather than single chromosome hybrids was revived by Walter and colleagues (1994) and has subsequently become a major tool for high-resolution mapping of the human genome. As reviewed by McCarthy (1996), panels of human radiation hybrids (RH) have been effectively utilized to integrate linkage and physical maps, to anchor or order large insert contigs, and to construct expressed sequence tag (EST) maps that already contain more than 12,000 entries and are growing rapidly. RH mapping has not been effectively utilized in constructing maps of other mammalian species, with the exception of the mouse (Schmitt et al. 1996). This situation is destined to change, however, owing to the potential of the technique for integrating linkage and physical maps. It is an especially powerful tool for comparative gene mapping, since chromosomal order can be established for expressed genes that are usually conserved between species but often recalcitrant to linkage mapping for lack of allelic variation. The bovine donor cell l used in constructing this panel were a normal diploid fibroblast culture established from an Angus bull, JEW38. Nearly confluent flasks were trypsinized and suspended in Gibco DMEM without supplements. Approximately 10 7 cells were irradiated at room temperature in 10 ml suspension medium in a T75 flask. A cobalt 60 source delivered 185 rad/min for a total dose of 5000 rad. Attached cells were removed with trypsin, and all cells were suspended in ca/mg-free Hanks balanced salt solution (HBSS), pH 8.0, at 106 cells/ml. One-half ml (5 x 105 cells) was removed as control and 4.5 ml used for fusion. The recipient Chinese hamster TKfibroblast line A23 was kindly provided by David Cox (Stanford University School of Medicine). These cells were also suspended in HBSS at 106/ml, and 0.7 ml of this suspension was removed as control and 9 ml used for fusion. JEW38 (4.5 x 10 6 cells) and A23 (9 x 10 6 cells) were thoroughly mixed, pelleted, and resuspended. One-half ml PEG (Boehringer Mannheim polyethylene glycol 1500 in 50% sterile solution) was slowly added with constant mixing. After 2 min, 10 ml HBSS, pH 8.0, was also added slowly with gentle mixing. Cells were pelleted, then resuspended in 5 ml HBSS, pH 8.0, for 15 min at 37~ Each control line was exposed to PEG by the same process. The fusion suspension was mixed into Gibco DMEM to 10% FBS plus HAT (Sigma) plus 5 x 10 -7 M ouabain to a total volume of 90 ml. Ten ml of this mixture was dispensed to each of nine 100-mm plates (approximately 1.5 x 105 cells/plate). Controls were mixed in the same solution and plated identically. All were incubated at 37~ in 5% CO 2. All JEW38 control cells were dead on day 7, while one A23 colony, apparently a TK revertant, sur-

Chromosome identification and assignment of DNA clones in the dog using a red fox and dog comparative map.

We have developed a novel method for identifying dog chromosomes and unambiguously mapping specific clones onto canine chromosomes. This method uses a previously established red fox/dog comparative chromosome map to guide the FISH mapping of cloned canine DNA. Mixing metaphase preparations of the red fox and dog enabled a single hybridization to be performed on both species. We used this approach to map the chromosomal locations of twenty-six canine cosmids. Each cosmid contains highly polymorphic microsatellite markers currently used by the DogMap project to compile the canine linkage map. All but two cosmids were successfully assigned to subchromosomal regions on red fox and dog chromosomes. For eight cosmids previously mapped on dog chromosomes, we confirmed and refined the canine chromosomal assignments of seven cosmids and corrected an erroneous assignment regarding cosmid CanBern1. These results demonstrate that the red fox and dog comparative chromosome map can greatly improve the accuracy and efficiency of chromosomal assignments of canine genetic markers by FISH.

A radiation hybrid framework map of bovine chromosome 13

In this paper we present a 5000-rad radiation hybrid framework map of bovine chromosome 13 (BTA13) containing 13 loci, including five conserved genes and eight polymorphic microsatellites. All framework markers are ordered with odds greater than 1000:1. Furthermore, we present a comprehensive map of BTA13 integrating 11 genes and 16 microsatellites. The proposed order is in general agreement with the recently published medium-density linkage maps. A model of five blocks of genes with conserved order between human, mouse and cattle is presented.

Development of a cytogenetic map for the Chinese raccoon dog (Nyctereutes procyonoides procyonoides) and the arctic fox (Alopex lagopus) genomes, using canine-derived microsatellite probes

New chromosomal assignments of canine-derived cosmid clones containing microsatellites to the Chinese raccoon dog and arctic fox genomes are presented in the study. The localizations are in agreement with data obtained from comparative chromosome painting experiments between the dog and arctic fox genomes. However, paracentric inversions have been detected by comparing the loci order in canid karyotypes. The number of physically mapped loci increased to thirty-five both in the Chinese raccoon dog and in the arctic fox. Furthermore, the present status of the cytogenetic map of the Chinese raccoon dog and arctic fox is presented in this study.

Twelve loci from HSA10, HSA11 and HSA20 were comparatively FISH-mapped on river buffalo and sheep chromosomes

Ten type I loci from HSA10 (IL2RA and VIM), HSA11 (HBB and FSHB) and HSA20 (THBD, AVP/OXT, GNAS1, HCK and TOP1) and two domestic cattle type II loci (CSSM30 and BL42) were FISH mapped to R-banded river buffalo (BBU) and sheep (OAR) chromosomes. IL2RA (HSA10) maps on BBU14q13 and OAR13q13, VIM (HSA10) maps on BBU14q15 and OAR13q15, HBB (HSA11) maps on BBU16q25 and OAR15q23, FSHB (HSA11) maps on BBU16q28 and OAR15q26, THBD (HSA20) maps on BBU14q15 and OAR13q15 while AVP/OXT, GNAS1, HCK, and TOP1 (HSA20) as well as CSSM30 and BL42 map on the same large band of BBU14q22 and OAR13q22. All loci were mapped on the same homologous chromosomes and chromosome bands of the two species, and these results agree with those earlier reported in cattle homologous chromosomes 15 and 13, respectively, confirming the high degree of both banding and physical map similarities among the bovid species. Indirect comparisons between physical maps achieved on bovid chromosomes and those reported on HSA10, HSA11 and HSA20 were performed.

Physical mapping of the endothelin receptor type B to bovine chromosome 12

kinase [1], is widely expressed in hematopoietic cells and is considered to be involved in the signal transductions from the B cell antigen receptor [4], high affinity IgE receptors [5] and T cell receptor [6]. The present study with FISH revealed that SYK gene resides on porcine Chr 14q14. A representative chromosome spread showing hybridization signals on Chr 14q14 was shown in Fig. 1. In this study, 142 chromosome spreads having signals were examined to locate the gene on chromosomes. One-hundred and thirty-two spreads of them showed signals on porcine Chr 14q14, whereas the rest showed no specific signal localization on porcine chromosomes. The ACTA1, ACTN2, LPL, ATA, PLAU, DAO, UBC, and GGT genes have been localized on porcine Chr 14 [7,8], where the SYK gene has been found in the present study. As for human chromosomes, the ACTA1 and ACTN2 genes are localized on Chr 1; LPL gene, on Chr 8; ATA and PLAU genes, on Chr 10; DAO and UBC genes, on Chr 12; and GGT gene, on Chr 22 [9]. Since the human SYK gene has been assigned to Chr 9q22 [3], our findings of SYK gene on porcine Chr 14q14 provide evidence that a part of porcine Chr 14, especially around q14 region, has homology with human Chr 9. Recently, ZOO-FISH analysis has been increasingly used to determine the correspondence of chromosomal regions between species. Rettenberger and associates [10] reported that porcine Chr 14 had homology with human Chrs 1, 8, 10, 12, and 22, whereas other groups reported that porcine Chr 14 had homology with human Chrs 8, 10, 12, and 22 [11,12]. The inconsistency of the correspondence between porcine Chr 14 and human Chr 1 in the reports is possibly owing to the limit of resolution in ZOO-FISH analysis. The correspondence between porcine Chr 14 and human Chr 9 observed in our study but not observed in the ZOO-FISH has revealed the limit of resolution in ZOO-FISH analysis. Therefore, admitting that the ZOO-FISH analysis is a useful tool for generating comparative maps, the precise comparative maps should be constructed by FISH analysis with individual DNA sequences as probes.

Genetic mapping of the bovine cytochrome c oxidase subunit IV pseudogene.

was recorded in the backcross progeny of 252 animals. Five polymorphic microsatellite markers were purchased from Research Genetics (MapPairs TM) and were designated by locus names as follows: D5MitlO, D5Mghl8, D5Mitll, D5Mghl7, and D5Mit7. The first four represent anonymous loci, whereas the last (D5Mit7) corresponds to the rat Slc2al gene. The marker genotypes could be resolved in all 252 backcross animals, and the MAPMAKER computer package [5] yielded the following average RNO5 linkage map (recombination fractions and standard errors in percentage format are given in parentheses): RNO5centromere-D5Mgh17(18.7 + 2.5)-D5MitlO-(16.3 +_ 2.3)-D5Mitl l-(6.8 +_ 1.6)-Tyrpl(17 .9 + 2 . 4 ) D 5 M i t 7 ( S l c 2 a l ) ( 5 . 2 +_ 1 . 4 ) D 5 M g h l 8 RNO5telomere. The Kosambi mapping function was used to calculate the cM distances (Fig. 1). The map covers about 70 cM of the chromosomes estimated total of about 120 cM (based on an assumed average genome length of 2000 cM in the rat). The order among the microsatellite markers was the same as that obtained by Jacob and associates [6] and by Truett and colleagues [7].