Divergence of Repetitive DNA Sequences in the Heterochromatin of Medaka Fishes: Molecular Cytogenetic Characterization of Constitutive Heterochromatin in Two Medaka Species: <b><i>Oryzias hubbsi</i></b> and <b><i>O. celebensis</i></b> (Adrianichthyidae, Beloniformes)

Abstract

The large biarmed chromosomes of Oryzias celebensis [2n = 36, fundamental arm number (FN) = 48] are considered to have resulted from fusions of acrocentric chromosomes in the ancestral karyotype of Oryzias, which is retained in O. hubbsi (2n = 48, FN = 48). To understand the molecular evolution of heterochromatin associated with karyotype reorganization in medaka fishes, we cloned 3 and 6 novel families of heterochromatin-related repetitive DNA sequences from O. hubbsi and O. celebensis, respectively, and characterized them using molecular cytogenetics. Two AT-rich repetitive sequences isolated from the genomic DNA of O. hubbsi, a 164-bp satellite DNA (OHU-RsaI-Scen) and a 177-bp telomere-specific repeat (OHU-RsaI-Stelo), were shown to be major components of the constitutive heterochromatin of centromeres and telomeres, respectively. A GC-rich 326-bp sequence, named OHU-AluI-M1, was colocalized with the 18S-28S ribosomal RNA gene cluster to a single autosomal pair of chromosomes and the W chromosome. In O. celebensis, 2 major satellite DNA sequences (the AT-rich 157-bp OCE-AluI-Scen sequence and the 186-bp OCE-HinfI-Scen sequence) were identified in the centromeric regions of almost all chromosomes. The 197-bp OCE-HinfI-S6 sequence was located in the centromeric and distal and/or interstitial heterochromatin of almost all chromosomes, and the 191-bp OCE-HinfI-S8 sequence was located in 6 pairs of chromosomes. Constitutive heterochromatin on the short arm of large submetacentric chromosome 5 was composed of at least 3 different repetitive sequences: the 171-bp OCE-AluI-S18 sequence, the 197-bp OCE-HinfI-S6 sequence and the 172-bp OCE-HinfI-S11 sequence. All families of repeated sequences showed no nucleotide sequence similarity with each other and high species-specificity among 7 different species. These results suggest that the heterochromatin of O. hubbsi and O. celebensis consists of various types of repetitive sequence and that the sequences evolved independently and were then amplified site-specifically in each lineage after karyotype reorganization occurred in the ancestral karyotype.

Molecular cytogenetic characterization of chromosome site-specific repetitive sequences in the Arctic lamprey (Lethenteron camtschaticum, Petromyzontidae)

“…Extraction of genomic DNA and molecular cloning of repetitive sequences were performed as previously described 22 . Genomic DNA was digested with restriction enzymes, electrophoresed on a 1.2% agarose gel, and DNA bands containing repetitive sequences were isolated from the gel and cloned into vectors.…”

Cell culture-based shark karyotyping as a resource for chromosome-scale genome analysis

“…To overcome this problem, we referred to past attempts and tried novel combinations of medium ingredients. For fibroblast culture, we derived culture medium supplemented with urea, NaCl and three kinds of cell growth factors (insulin-transferrin-selenium [ITS-G], epidermal growth factor and fibroblast growth factor) at the concentrations used in previous studies 22,24,28,30 ( Supplementary Fig. 1, Supplementary Table 2).…”

“…The whole embryos and tissues were washed in Dulbecco's modified Eagle's medium (Thermo Fisher Scientific-GIBCO, Carlsbad, CA, USA) containing a high concentration (5%) of antibiotic-antimycotic solution (Thermo Fisher Scientific-GIBCO), 373 mM urea and 89 mM NaCl with the pH adjusted to 7.3 25 . The washed whole embryos and tissues were minced with sterilized scissors and plated on a collagen I-coated culture dish (AGC Techno Glass, Shizuoka, Japan), and cultured in LDF medium, a mixture of 50% Dulbecco's modified Eagle's medium, 35% L-15 and 15% Ham's F-12, supplemented with 12% foetal bovine serum (FBS), 1% antibiotic-antimycotic solution, 1% ITS-G, 100 μg/ml kanamycin, 2 ng/ml epidermal growth factor, 2 ng/ml fibroblast growth factor (all from Thermo Fisher Scientific-GIBCO), 333 mM urea, 188 mM NaCl and 54 mM trimethylamine N-oxide, with the pH adjusted to 7.3 22,24,28,30 . The cultures were incubated at 26 °C in a humidified atmosphere of 5% CO 2 .…”

“…Fluorescence in situ hybridization. Fluorescence in situ hybridization (FISH) analysis was performed as described previously 30,56 . To determine the chromosomal location of the 18S-28S rRNA genes, we used pHr21Ab (5.8 kb for the 5′ portion) and pHr14E3 (7.3 kb for the 3′ portion) fragments of the human 45S pre-ribosomal RNA gene (RNA45S), which encodes a precursor RNA for 18S, 5.8S and 28S rRNAs, as the FISH probe as in our previous studies 17,18,30 .…”

“…Multipassage fibroblast culture for cartilaginous fish has been documented only for the spiny dogfish shark Squalus acanthias (later designated Squalus suckleyi), in which the addition of cell growth factors and shark yolk extract allowed a continuously proliferating cell line 28 . These growth factors have also been used for fibroblast culture of teleost fishes 29,30 . For lymphocyte culture, mitogens are the most important factors affecting the mitotic index and trigger a polyclonal proliferation of lymphocytes through blastoid transformation.…”