Mapping of rRNA Gene Loci in the Mice, Mus musculus molossinus (Japan) and Mus musculus musculus (Russia) by Double Color FISH

Abstract

ABSTRACT. Ribosomal RNA gene (rDNA) loci of Russian Mus musculus musculus and of Japanese Mus musuculus molossinus were mapped by double color FISH. The total number of rDNA loci was varied from 5 to 12, although the loci on chromosomes 12, 15, 16, 18, and 19 were common to all mice examined. Instead, polymorphisms of the rDNA loci were found on chromosomes 1, 5, 8, 9, 10, 11, 13 and 17 [4,14]. The karyotype of these subspecies is identical except for the difference in sizes of the Y chromosome [10]. In the laboratory strains, the total number of rDNA loci varied from three to five, and the loci were limited to chromosomes 12, 15, 16, 18 and 19 [1, 3, 5, 9]. Although the locus polymorphism was found on chromosomes 12, 15, 16 and 19, rDNA locus on the chromosome 18 was observed consistently in all laboratory strains examined. Such variations of rDNA loci on these chromosomes were reported in the wild mice of Mus musculus species, as well as the polymorphism of rDNA loci on other chromosomes [11,13].The evolution of rDNA has been the subject of controversy for many years, as the clusters of rDNA at different chromosomal loci retain the strict sequence homology and the copy number of the gene varied about one to several hundred in mammals [12]. Mus musculus species is a good model system for the analysis of rDNA evolution, because of the remarkable variation of rDNA loci not only between different subspecies but also within the same subspecies. In the present study, to elucidate the evolution of rDNA loci in Mus musculus, we further determined rDNA loci in wild and wild mouse-derived strains of M. m. molossinus and wild mouse-derived strains of M. m. musculus.A wild M. m. molossinus was captured in Machida, Tokyo, Japan. The JF1 strain of M. m. molossinus was derived from fancy mice and established as a strain at the National Institute of Genetics, Mishima, Japan [8]. Three wild mouse strains of M. m. musculus were derived from Okha, Irkutsk and Novokachalinsk in Russia and established by one of authors (T. K.) as inbred strains Okh/TUA, Irk/TUA and Nov/TUA, respectively. Lymphocytes, isolated from the spleen, were cultured, and air-dried chromosome slides were prepared following the protocol of Matsuda et al. [11].A human 28S-rDNA (pHr14E3, Human Science Research Resource Bank, Japan) was labeled by nick translation with digoxigenin-11-dUTP and ethanol-precipitated with salmon sperm DNA and tRNA. This probe DNA was dissolved in 8 µl of 100% formamide and denatured at 75°C for 10 min. Biotin-labeled mouse chromosome paints (Cambio, Cambridge, UK) were denatured following the manufacturer's protocol. The labeled probes, which consist of 8 µl of rDNA and 3 µl of chromosome paints, were mixed with 11 µl of hybridization buffer (10 × SSC, 1 mg/ml BSA and 50% dextran sulfate). Prior to hybridization, the slides were treated with RNase (100 µg/ml in 2 × SSC) for 1 hr to remove the cellular rRNA. The slides were washed in 2 × SSC, dehydrated in an ethanol series, denatured at 70°C for 2 min in 70% formamide in 2 × SSC...

Chromosomal dynamics of nucleolar organizer regions (NORs) in the house mouse: micro-evolutionary insights

“…A total of 16 NOR-bearing chromosomes were identified comprising 16 centromeric and 2 telomeric locations on these NOR dynamics in the house mouse J Britton-Davidian et al chromosome pairs. All of these NOR sites (except NOR 14) have previously been identified in the species (Winking et al, 1980;Suzuki et al, 1990;Ramalhinho et al, 2005;Ito et al, 2007Ito et al, , 2008, but their subspecific distribution is here refined (for example, NORs 8, 9, 10 and 17 were previously unknown in DOM). The frequency distribution of NORs showed considerable variation between chromosome pairs within and between subspecies.…”

“…Four of the five subspecies of M. musculus are represented in this survey: M. m. domesticus (DOM), M. m. musculus (MUS), M. m. castaneus (CAS), and M. m. gentilulus (GEN). The data pertaining to the fifth subspecies, M. musculus molossinus (MOL) were sampled from the literature (Suzuki et al, 1990;Ito et al, 2007Ito et al, , 2008. DOM individuals carrying Robertsonian fusions (see Piálek et al, 2005) were included in the analyses to increase the geographical coverage of this subspecies (see Supplementary Table S1).…”

“…The following nomenclatures were adopted: NOR followed by a number designates the NOR-bearing chromosome; telomeric NORs are indicated by a T after the chromosome number; a NOR site refers to the location of the rRNA genes on a single chromosome. The data were completed with those available in the literature for wild individuals of DOM, MUS, CAS and MOL, using either the FISH approach (Fel-Clair et al, 1998;Ito et al, 2007Ito et al, , 2008Cazaux et al, 2011), or the Ag-NOR staining method when genotype scores could be extracted (Winking et al, 1980;Suzuki et al, 1990;Ramalhinho et al, 2005). Details on the procedure used for genotype scoring are indicated in Supplementary Table S1.…”

Polymorphism of rRNA Gene Loci in the Dog

“…The polymorphisms of the rDNA loci were reported in a variety of species, especially in those with the rDNA loci located on the acrocentric chromosomes such as in the mouse [1,9,11,15] and humans [7,8]. In the dog, rDNA loci were mapped to the terminal ends of the long arms of three autosomes (#7, #11, and #27) and to the terminal end of the short arm of the Y chromosome by AgNOR method (10), or to those of chromosome #7, #9 and #22 by in situ hybridization autoradiography [6].…”

“…Air-dried chromosome preparations were dried at 37°C for a few days in an incubator. For FISH analysis, chromosomal DNA was denatured in 70% formamide in 2× SSC at 70°C for 2 min and hybridized with the biotin labeled rDNA as described previously [9]. Human 28S-rDNA clone [pHr14E3, Human Science Research Resourse Bank (HSRRB), Japan] was labeled with biotin-dUTP by nick translation and denatured in formamide at 70°C, and then hybridized to the denatured chromosomal DNA at 37°C overnight.…”