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

Abstract

All extant lamprey karyotypes are characterized by almost all dot-shaped microchromosomes. To understand the molecular basis of chromosome structure in lampreys, we performed chromosome C-banding and silver staining and chromosome mapping of the 18S–28S and 5S ribosomal RNA (rRNA) genes and telomeric TTAGGG repeats in the Arctic lamprey (Lethenteron camtschaticum). In addition, we cloned chromosome site-specific repetitive DNA sequences and characterized them by nucleotide sequencing, chromosome in situ hybridization, and filter hybridization. Three types of repetitive sequences were detected; a 200-bp AT-rich repetitive sequence, LCA-EcoRIa that co-localized with the 18S–28S rRNA gene clusters of 3 chromosomal pairs; a 364-bp AT-rich LCA-EcoRIb sequence that showed homology to the EcoRI sequence family from the sea lamprey (Petromyzon marinus), which contains short repeats as centromeric motifs; and a GC-rich 702-bp LCA-ApaI sequence that was distributed on nearly all chromosomes and showed significant homology with the integrase-coding region of a Ty3/Gypsy family long terminal repeat (LTR) retrotransposon. All three repetitive sequences are highly conserved within the Petromyzontidae or within Petromyzontidae and Mordaciidae. Molecular cytogenetic characterization of these site-specific repeats showed that they may be correlated with programed genome rearrangement (LCA-EcoRIa), centromere structure and function (LCA-EcoRIb), and site-specific amplification of LTR retroelements through homogenization between non-homologous chromosomes (LCA-ApaI).

Cell culture-based karyotyping of orectolobiform sharks for chromosome-scale genome analysis

“…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 19 , 20 , 31 . The DNA fragments, which were provided by National Institutes of Biomedical Innovation, Health and Nutrition, Osaka, were labelled with biotin 16-dUTP using a nick translation kit (Roche Diagnostics, Basel, Switzerland) following the manufacturer’s instructions, and ethanol precipitated with salmon sperm DNA and Escherichia coli RNA (all from Sigma-Aldrich), followed by denaturation at 75 °C for 10 min in 100% formamide 31 , 57 .…”

“…Cell culture is an important tool for studies using traditional laboratory animals such as the mouse and chicken. When applied to chromosome studies, cell culture facilitates the preparation of high-quality chromosome spreads with high metaphase frequency, as exemplified by the karyotyping of diverse species, including one with more than 150 chromosomes 19 , and by high-throughput chromosome mapping using fluorescence in situ hybridization (FISH) 20 . However, it has remained to be stably applied to chondrichthyans whose blood osmolality is approximately three times higher than that of mammalians and teleost fishes 8 .…”

Why Do Some Vertebrates Have Microchromosomes?

“…), racerunner lizard ( Eremias spp. ), and snake-eyed lizard ( Ophisops elegans ) to more than 40 pairs in Arctic lamprey ( Lethenteron camtschaticum ) [ 28 , 55 , 56 , 57 , 58 ] ( Figure 1 ). The occurrence of a diversity in chromosome number between different vertebrates presents an opportunity to correlate chromosome evolution with the timing and types of events [ 27 ].…”

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

“…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 . The DNA fragments, which were provided by National Institutes of Biomedical Innovation, Health and Nutrition, Osaka, were labelled with biotin 16-dUTP using a nick translation kit (Roche Diagnostics, Basel, Switzerland) and hybridized to metaphase chromosomes overnight at 37 °C.…”

“…Cell culture is an important tool for studies using traditional laboratory animals such as the mouse and chicken. When applied to chromosome studies, cell culture facilitates the preparation of high-quality chromosome spreads with high metaphase frequency, as exemplified by the karyotyping of diverse species, including one with more than 150 chromosomes 17 , and by high-throughput chromosome mapping using fluorescence in situ hybridization (FISH) 18 . However, it has remained to be stably applied to chondrichthyans whose blood osmolality is approximately three times higher than that of mammalians and teleost fishes 19 .…”