Whole-genome radiation hybrid (RH) mapping has proven to be a powerful tool for mapping genes and comparing genome architecture. We describe a protocol for constructing RH panels by rescuing irradiated fibroblast donor cells of any mammalian species by polyethylene glycol fusion to a thymidine kinase-deficient hamster cell line. Characterization and expansion of a panel of 90-100 cell lines can be used to map virtually any PCR-based marker that can be distinguished from the recipient hamster genome. The described procedure has been used successfully to create RH panels from diverse mammalian species such as macaques, elephants, alpacas, and armadillos, and may be applicable to nonmammalian vertebrates as well.

Among the 37 living species of Felidae, the clouded leopard (Neofelis nebulosa) is generally classified as a monotypic genus basal to the Panthera lineage of great cats. This secretive, mid-sized (16-23 kg) carnivore, now severely endangered, is traditionally subdivided into four southeast Asian subspecies (Figure 1A). We used molecular genetic methods to re-evaluate subspecies partitions and to quantify patterns of population genetic variation among 109 clouded leopards of known geographic origin (Figure 1A, Tables S1 ans S2 in the Supplemental Data available online). We found strong phylogeographic monophyly and large genetic distances between N. n. nebulosa (mainland) and N. n. diardi (Borneo; n = 3 individuals) with mtDNA (771 bp), nuclear DNA (3100 bp), and 51 microsatellite loci. Thirty-six fixed mitochondrial and nuclear nucleotide differences and 20 microsatellite loci with nonoverlapping allele-size ranges distinguished N. n. nebulosa from N. n. diardi. Along with fixed subspecies-specific chromosomal differences, this degree of differentiation is equivalent to, or greater than, comparable measures among five recognized Panthera species (lion, tiger, leopard, jaguar, and snow leopard). These distinctions increase the urgency of clouded leopard conservation efforts, and if affirmed by morphological analysis and wider sampling of N. n. diardi in Borneo and Sumatra, would support reclassification of N. n. diardi as a new species (Neofelis diardi).

We studied the chromosomes of an Afrotherian species, the short-eared elephant shrew Macroscelides proboscideus with traditional banding techniques and mapped the homology to human chromosomes by in-situ hybridization of human chromosome paints. Here we present for the first time the karyotype of this species, including banding patterns. The chromosome painting allowed us to test various hypotheses of the ancestral Eutherian karyotype, the validity of the radical taxonomic assemblage known as Afrotheria and the phylogenetic position of the elephant shrew within the Afrotheria. Current hypotheses concerning the Eutherian ancestral karyotype include diploid numbers ranging from 2n = 44 to 50 while molecular studies have proposed a new superordinal grouping of extant Eutherians. In particular, the Afrotheria is hotly debated, as it appears to be an odd mixture of species from Ungulata, Tubulidentata, Macroscelidea and Lipotyphla, which have no apparent morphological traits to unite them. The hybridization pattern delimited a total of 37 segments in the elephant shrew genome and revealed 21 different associations of human chromosome segments. Associations 1/19 and 5/21 link all Afrotheria so far studied and support the Afrotheria assemblage. Associations 2/8, 3/20, and 10/17 strongly link aardvarks and elephant shrews after the divergence of the line leading to elephants. The most likely ancestral Eutherian karyotype would be 2n = 48 chromosomes. However, the lack of comparative chromosome painting data between Eutherians and an appropriate outgroup is a severe limitation on attempts to delineate the ancestral genome of Eutherians. Current attempts lack legitimacy until this situation is corrected.

To investigate the mutagenic consequences of BP-DNA adduct formation, oligonucleotides, containing a single purine adduct derived from trans opening of each of the four BPDE configurational isomers, were ligated into M13mp7L2 and then transfected into E. coli SMH77 largely as described by Lawrence et al., Nucleic Acids Res. 18, 2153 (1990). Each adduct was placed in sequence contexts corresponding to mutation hotspots of coldspots in the supF gene of pS189. Replication efficiency, relative to phage containing unadducted oligonucleotide, indicated that dAdo adducts in a hotspot context were the most effective blocks to replication and that SOS induction increased progeny from dAdo adduct-laden phage more than from dGuo adduct-laden phage. Differences in adduct configuration also had substantial effects (up to 20-fold) on replication efficiency. When screened with probes for wild-type sequence, plaques from unadducted oligonucleotides were predominantly positive, those from dAdo adducts in the hotspot context were negative, and while some from dGuo adducts were negative, most were positive. Both adduct configuration and sequence context were important determinants of replication efficiency.