Ricky Critcher

A radiation hybrid map of the rat genome containing 5,255 markers

A whole-genome radiation hybrid (RH) panel was used to construct a high-resolution map of the rat genome based on microsatellite and gene markers. These include 3,019 new microsatellite markers described here for the first time and 1,714 microsatellite markers with known genetic locations, allowing comparison and integration of maps from different sources. A robust RH framework map containing 1,030 positions ordered with odds of at least 1,000:1 has been defined as a tool for mapping these markers, and for future RH mapping in the rat. More than 500 genes which have been mapped in mouse and/or human were localized with respect to the rat RH framework, allowing the construction of detailed rat-mouse and rat-human comparative maps and illustrating the power of the RH approach for comparative mapping.

A bovine whole-genome radiation hybrid panel and outline map

A 3000-rad radiation hybrid panel was constructed for cattle and used to build outline RH maps for all 29 autosomes and the X and Y chromosomes. These outline maps contain about 1200 markers, most of which are anonymous microsatellite loci. Comparisons between the RH chromosome maps, other published RH maps, and linkage maps allow regions of chromosomes that are poorly mapped or that have sparse marker coverage to be identified. In some cases, mapping ambiguities can be resolved. The RH maps presented here are the starting point for mapping additional loci, in particular genes and ESTs that will allow detailed comparative maps between cattle and other species to be constructed. Radiation hybrid cell panels allow high-density genetic maps to be constructed, with the advantage over linkage mapping that markers do not need to be polymorphic. A large quantity of DNA has been prepared from the cells forming the RH panel reported here and is publicly available for mapping large numbers of loci.

Co‐localization of centromere activity, proteins and topoisomerase II within a subdomain of the major human X α‐satellite array

Dissection of human centromeres is difficult because of the lack of landmarks within highly repeated DNA. We have systematically manipulated a single human X centromere generating a large series of deletion derivatives, which have been examined at four levels: linear DNA structure; the distribution of constitutive centromere proteins; topoisomerase IIα cleavage activity; and mitotic stability. We have determined that the human X major α‐satellite locus, DXZ1, is asymmetrically organized with an active subdomain anchored ∼150 kb in from the Xp‐edge. We demonstrate a major site of topoisomerase II cleavage within this domain that can shift if juxtaposed with a telomere, suggesting that this enzyme recognizes an epigenetic determinant within the DXZ1 chromatin. The observation that the only part of the DXZ1 locus shared by all deletion derivatives is a highly restricted region of <50 kb, which coincides with the topo isomerase II cleavage site, together with the high levels of cleavage detected, identify topoisomerase II as a major player in centromere biology.

Molecular analysis of 9p deletions associated with XY sex reversal: refining the localization of a sex-determining gene to the tip of the chromosome.

We thank Dr. C. P. Bennet, R. Hawkins, and Dr. O. Zuffardi, for providing us with some of the patient material; Giuliana Peverali, for technical support in the microsatellite typing; M. Davies, for technical support in tissue culture; and Drs. C. Farr and E.A. Oakenfull, for critical reading of the manuscript. This work was supported by Welcome grant 035201/Z/92 (to P.N.G and S.G.), EU grant Biomed2-CT960790 (to P.N.G.), a Telethon research fellowship (to S.G.), and an EMBO long-term fellowship (to K.S.).

Construction and characterization of zebrafish whole genome radiation hybrids.

Publisher Summary This chapter describes the construction and characterization of zebrafish whole genome radation hybrids. Zebrafish whole genome radiation hybrids are produced by fusing irradiated zebrafish cells to a hamster cell line that is hypoxanthine phosphoribosyl transferase (HPRT) or thymidine kinase (TK) deficient. The irradiation procedure randomly breaks the zebrafish genome, and the DNA fragments generated are rescued via the fusion process. Surviving cells are grown in hypoxanthine-aminopterinethymidine (HAT) medium, which selects for hybrids containing the zebrafish HPRT gene or TK gene. After picking the surviving hybrid clones and DNA extraction, the panel of hybrids is tested by polymerase chain reaction (PCR) for retention of genomic fragments by typing several microsatellite markers across the genome. For large-scale mapping or positional cloning projects, statistical programs are available to calculate map order and distances based on PCR typing of many markers across the hybrid panel. The advantages of whole genome radiation hybrids are (1) the ability to map any marker as long as it differs between rodent and zebrafish and (2) tailoring panels to different mapping resolutions depending on the application by adjusting the radiation dose.

A horse whole-genome-radiation hybrid panel: chromosome 1 and 10 preliminary maps.

In recent years there has been increasing interest in mapping thehorse genome, particularly to identify disease and performance-enhancing genes. Although a number of horse mapping tools havebeen developed and have proved very useful (genetic maps, so-matic cell hybrid panels, and a BAC library), the benefits ofwhole-genome–radiation hybrid (WG-RH) mapping have not beenavailable. Its advantages over genetic linkage mapping are: (i) theresolving power is not lost in regions of the genome with a lowrecombination rate, because it does not rely on meiotic recombi-nation events; (ii) it is especially useful in animals such as thehorse with relatively long generation times and single births; and(iii) genetic and physical maps can be integrated, as both poly-morphic and non-polymorphic markers can be placed on the samemap. The WG-RH panel constructed in this study is the first suchpanel to be reported for the horse, and its preliminary character-ization demonstrates its usefulness for horse genome mapping.The panel was constructed by the fusion of horse embryonicendothelial primary lung cells (male) to the established hamsterfibroblast cell line A23 (Westerveld et al. 1971) by using themethod described in McCarthy et al. (1997). A series of fusions,involving irradiation (3000 rads) of donor cells prior to fusion withequal numbers of recipient cells, generated ∼160 hybrids in total.From these 160 hybrids, 94 were selected at random and screenedby using 20 widely distributed markers (representing 17 chromo-somes). Any hybrids that did not produce an amplification productwith these markers were screened by FISH to determine whetherthey contained horse DNA from other chromosomes or regions ofchromosomes (not represented by the 20 markers); hybrids foundto be negative by the FISH screen were replaced at random fromthe remaining unused hybrids, and the same PCR and FISH screen-ing procedure was repeated until 94 hybrids were assembled.These 94 hybrids were used for preliminary characterization as amapping panel. It is expected that the majority of the hybrids(>95%) characterized here will be represented in the TM99 panelof 94 hybrids that is being grown on a large scale by ResearchGenetics Inc. (Huntsville, Ala. 35801).The amount of horse DNA retained by the hybrids in the panelwas evaluated by examining the retention of the 20 widely dis-tributed horse markers. On average each marker was retained in27.8% of the hybrids (ranging from 10.6% to 71.3%; Table 1),implying that the panel as a whole retains the equivalent of ap-proximately 26 horse genomes. These retention frequencies com-pare well with those found for the human and mouse RH panels,which have been used successfully for creating whole-genomemaps (Gyapay et al. 1996; McCarthy et al. 1997).The mapping ability of the panel was assessed by producingRH maps for the two horse chromosomes with the most markersavailable, and comparing these with the latest genetic maps (Swin-burne et al. 2000a). In total, 39 markers on Chromosome (Chr) 1and 15 markers on Chr 10 were analyzed (Table 1). The averageretention of markers was 15.4% (ranging from 5.3% to 29.8%) onChr 1, and was higher, 25.4% (ranging from 16.0% to 44.7%) onChr 10. An increase in the retention frequency on smaller chro-mosomes was also noted in human and mouse RH panels (Gyapayet al. 1996; McCarthy et al. 1997). For each chromosome, linkagegroups with at least 4-LOD units support were identified; four suchgroups were found on Chr 1 and two were detected on Chr 10(Figs. 1 and 2). Within each of these linkage groups, frameworkmarkers were ordered with 3-LOD units support, and most of thenon-framework markers were ordered with 2-LOD units support.Some non-framework markers had lower statistical support fortheir order (shown in italics, Figs. 1 and 2). Similarly, the relativeorder of some linkage groups was suggested by linkage analysisbut had low statistical support [Chr 1 (groups B and C) and Chr 10(groups A and B)]. The relative order of the other RH linkagegroups was determined by comparison with the genetic map andFISH localizations.The genetic map can give a misleading impression of markerdensity because genetic distances may be small owing to regionshaving a low meiotic recombination rate. In such regions the mark-ers may actually be physically far apart and, therefore, at a lowerdensity than predicted by the genetic map. Since RH panels requirea high density of markers, this might account for the low statisticalsupport for the ordering of some markers and linkage groups. Alow density of available markers could also explain why two mark-ers (HLM5, ICA22) could not be placed on the RH map. HLM5 hasalready been shown to be 24 cM from its nearest neighboringmarker on the genetic map. Three other markers could not beplaced on the RH map (UM004, HTG12, and HMS15), but wereable to be placed on the genetic map in a region equivalent to RHlinkage group D; this discrepancy between the two maps should beresolved as both maps are characterized further.This study has demonstrated the ability of the horse WG-RHpanel to produce an accurate genome map as there is good agree-ment of the genetic and physical maps with the RH maps for Chrs1 and 10 (Figs. 1 and 2). Only a few differences between the mapswere observed, and experiences with human mapping suggest thatsuch differences are common and are resolved as more markers areincorporated into the maps (Walter et al. 1994). The WG-RH panelwas able to order some markers that co-segregate on the geneticlinkage map, i.e., Chr 1 markers LEX39 and ICA18, and ICA41 andICA32, and Chr 10 markers LEX8 and COR015, and NVHEQ18

A whole-genome radiation hybrid panel and framework map of the rat genome

Linda C. McCarthy, * ** Marie-Therese Bihoreau,* Susanna L. Kiguwa,* Julie Browne, Takeshi K. Watanabe, Haretsugu Hishigaki, Atsushi Tsuji, Susanne Kiel, 2 Caleb Webber, Maria E. Davis, Catherine Knights, Angela Smith, Ricky Critcher, 1 Patrick Huxtall, 1 James R. Hudson, Jr., 4 Toshihide Ono, Hiroumi Hayashi, Toshihisa Takagi, Yusuke Nakamura, Akira Tanigami, 3 Peter N. Goodfellow, *** G. Mark Lathrop, 2 Michael R. James

Genomic characterisation and fine mapping of the human SOX13 gene.

SOX13 is the member of the SOX (Sry related HMG BOX) family of transcription factors which encodes the type-1 diabetes autoantigen, ICA12, and is expressed in a number of tissues including pancreatic islets and arterial walls. By fluorescence in situ hybridisation, radiation hybrid mapping and YAC analysis we determined that the human SOX13 gene maps to Chromosome 1q31.3-32.1 near the marker D1S504, a region associated with type-1 diabetes susceptibility and familial dilated cardiomyopathy. Mouse Sox13 maps to the syntenic region near the marker D1Mit57. The human SOX13 gene spans >15.5kb of genomic DNA and is composed of 14 exons with introns interrupting regions encoding the HMG DNA binding domain and the leucine zipper/glutamine-rich dimerisation domain. Comparison with the mouse Sox13 gene suggests the existence of long and short forms of the SOX13 protein which may arise by differential splicing during different stages in embryogenesis. The high sequence conservation between human SOX13 and mouse, Xenopus and trout orthologues implies a conserved function in vertebrates. SOX13 belongs to SOX Group D members which contain a leucine zipper/glutamine-rich region. Phylogenetic analyses of SOX proteins suggest that such domains were acquired after the initial divergence of groups A to G.