, Volume 102, Issue 3, pp 163–173 | Cite as

Germ line-restricted, highly repeated DNA sequences and their chromosomal localization in a Japanese hagfish (Eptatretus okinoseanus)

  • Souichirou Kubota
  • Masaki Kuro-o
  • Shigeki Mizuno
  • Sei-ichi Kohno
Original Articles


The various species of Japanese hagfish, namely, Eptatretus okinoseanus (types A and B), Eptatretus burgeri and Myxine garmani, are known to eliminate a fraction of their chromosomes during early embryogenesis. High molecular weight DNA from germ line cells and somatic cells of these hagfish species was isolated and digested with different restriction enzymes. The DNA fragments were separated by agarose gel electrophoresis. Digestion with BamHI and DraI generated two weak bands and one weak band, respectively, that were estimated to be about 90, and 180 bp and about 90 bp long and were limited to the germ line DNA in both types of E. okinoseanus. DNA filter hybridization experiments showed that the two BamHI fragments and the one DraI fragment were present almost exclusively in the germ line DNA of E. okinoseanus. Thus, these DNA fragments appear to be eliminated during embryogenesis. Moreover, evidence was obtained that these fragments are highly and tandemly repeated. Molecular cloning and sequence analysis revealed that the BamHI fragments are mainly composed of a family of closely related sequences that are 95 bp long (EEEo1, for Eliminated Element of E. okinoseanus 1), and the DraI fragment is composed of another family of closely related sequences that are 85 bp long (EEEo2). The two DNA families account for about 19% of the total eliminated DNA in E. okinoseanus type A. Fluorescence in situ hybridization experiments demonstrated that the two families of DNA are located on several C-band-positive, small chromosomes that are limited to germ cells in both types of E. okinoseanus.


Germ Cell Chromosomal Localization Germ Line Weak Band Hybridization Experiment 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. AebyP, SpicherA, deChastonayY, MüllerF, ToblerH (1986) Structure and genomic organization of proretrovirus-like elements partially eliminated from the somatic genome of Ascaris lumbricoides. EMBO J 5: 3353–3360PubMedGoogle Scholar
  2. AmmermannD (1985) Chromatin diminution and chromosome elimination: mechanisms and adaptive significance. In: Cavalier-SmithT (ed) The evolution of genome size. Wiley, New York, pp 427–442Google Scholar
  3. AndersonMLM, YoungBD (1985) Quantitative filter hybridisation. In: HamesBD, HigginsSJ (ed) Nucleic acid hybridisation. IRL, Oxford, pp 73–111Google Scholar
  4. BostockC (1971) Repetitious DNA. Adv Cell Biol 2: 153–223PubMedGoogle Scholar
  5. BoveriT (1887) Über Differenzierung der Zellkerne während der Furchung des Eies von Ascaris megalocephala. Anat Anz 2: 688–693Google Scholar
  6. BrutlagDL (1980) Molecular arrangement and evolution of heterochromatic DNA. Annu Rev Genet 14: 121–144CrossRefPubMedGoogle Scholar
  7. ChomczynskiP, QasbaPK (1984) Alkaline transfer of DNA to plastic membrane. Biochem Biophys Res Commun 122: 340–344PubMedGoogle Scholar
  8. DenhardtDT (1966) A membrane-filter technique for the detection of complementary DNA. Biochem Biophys Res Commun 23: 641–646PubMedGoogle Scholar
  9. Etter A, Spicher A, Aboutanos M, Müller F, Tobler H (1990) A gene contained within the eliminated chromatin of Ascaris lumbricoides. Experientia 46:A22Google Scholar
  10. EtterA, AboutanosM, ToblerH, MüllerF (1991) Eliminated chromatin of Ascaris contains a gene that encodes a putative ribosomal protein. Proc Natl Acad Sci USA 88: 1593–1596PubMedGoogle Scholar
  11. FlammG (1972) Highly repetitive sequences of DNA in chromosomes. Int Rev Cytol 32: 1–51PubMedGoogle Scholar
  12. GallJG, PardueML (1971) Nucleic acid hybridization in cytological preparations. Methods Enzymol 21: 470–480Google Scholar
  13. HattoriM, SakakiY (1986) Dideoxy sequencing method using denatured plasmid templates. Anal Biochem 152: 232–238PubMedGoogle Scholar
  14. HennigW (1973) Molecular hybridization of DNA and RNA insitu. Int Rev Cytol 36: 1–44PubMedGoogle Scholar
  15. HennigW (1986) Heterochromatin and germ line-restricted DNA. In: HennigW (ed) Germ line-soma differentiation. (Results and problems in cell differentiation 13) Springer, Berlin Heidelberg, pp 175–192Google Scholar
  16. HennigW, HennigI, SteinH (1970) Repeated sequences in DNA of Drosophila and their localization in giant chromosomes. Chromosoma 32: 31–63PubMedGoogle Scholar
  17. JohnB, MiklosGLG (1979) Functional aspects of satellite DNA and heterochromatin. Int Rev Cytol 58: 1–114PubMedGoogle Scholar
  18. KirbyKS (1956) A new method for the isolation of ribonucleic acid from mammalian tissues. Biochem J 64: 405–408PubMedGoogle Scholar
  19. KohnoS, NakaiY, SatohS, YoshidaM, KobayashiH (1986) Chromosome elimination in Japanese hagfish, Eptatretus burgeri (Agnatha, Cyclostomata). Cytogenet Cell Genet 41: 209–214PubMedGoogle Scholar
  20. KönenM (1989) Recovery of DNA from agarose gels using liquid nitrogen. Trends Genet 5: 137CrossRefGoogle Scholar
  21. KramR, BotchanM, HearstJE (1972) Arragement of highly reiterated DNA sequences in the centrometric heterochromatin of Drosophila melanogaster: Evidence for interspersed spacer DNA. J Mol Biol 64: 103–117PubMedGoogle Scholar
  22. KubotaS, NakaiY, Kuro-oM, KohnoS (1992) Germ line-restricted supernumerary (B) chromosomes in Eptatretus okinoseanus. Cytogenet Cell Genet 60: 224–228PubMedGoogle Scholar
  23. LawrenceJB, VillnaveCA, SingerRH (1988) Sensitive, high-resolution chromatin and chromosome mapping in situ: Presence and orientation of two closely integrated copies of EBV in a lymphoma line. Cell 52: 51–61CrossRefPubMedGoogle Scholar
  24. McClellandM, NelsonM (1988) The effects of site-specific DNA methylation on restriction endonucleases and DNA modification methyltransferases. Gene 74: 291–304CrossRefPubMedGoogle Scholar
  25. MüllerF, WalkerP, AebyP, NeuhausH, BackE, ToblerH (1982a) Molecular cloning and sequence analysis of highly repetitive DNA sequences contained in the eliminated gonome of Ascaris lumbricoides. In: BurgerMM, WeberR (eds) Embryonic development, part A: Genetic aspects. Liss, New York, pp 127–138Google Scholar
  26. MüllerF, WalkerP, AebyP, NeuhausH, FelderH, BackE, ToblerH (1982b) Nucleotide sequence of satellite DNA contained in the eliminated genome of Ascaris lumbricoides. Nucleic Acids Res 10: 7493–7510PubMedGoogle Scholar
  27. Nakai Y (1987) Chromosome elimination in four Japanese hagfishes, Eptatretus burgeri, Eptatretus okinoseanus, Paramyxine atami and Myxine garmani. Thesis for Master of Science degree, Toho University, JapanGoogle Scholar
  28. NakaiY, KohnoS (1987) Elimination of the largest chromosome pair during differentiation into somatic cells in Japanese hagfish, Myxine garmani (Cyclostomata, Agnatha). Cytogenet Cell Genet 45: 80–83Google Scholar
  29. NakaiY, KubotaS, KohnoS (1991) Chromatin diminution and chromosome elimination in four Japanese hagfish species. Cytogenet Cell Genet 56: 196–198PubMedGoogle Scholar
  30. Ojima Y (1983) Fish cytogenetics (in Japanese). Suikohsha, TokyoGoogle Scholar
  31. PimpinelliS, GodayC (1989) Unusual kinetochores and chromatin diminution in Parascaris. Trends Genet 5: 310–315CrossRefPubMedGoogle Scholar
  32. PimpinelliS, BonaccorsiS, GattiM, SandlerL (1986) The peculiar genetic organization of Drosophila heterochromatin. Trends Genet 2: 17–20CrossRefGoogle Scholar
  33. RaePMM (1972) The distribution of repetitive DNA sequences in chromosomes. Adv Cell Mol Biol 2: 109–149Google Scholar
  34. SambrookJ, FritschEF, ManiatisT (1989) Molecular cloning. A laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  35. SangerF, NicklenS, CoulsonAR (1977) DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74: 5463–5467PubMedGoogle Scholar
  36. SingerMF (1982) Highly repeated sequences in mammalian genomes. Int Rev Cytol 76: 67–112PubMedGoogle Scholar
  37. SouthernEM (1970) Base sequence and evolution of guinea pig satellite DNA. Nature 227: 794–798PubMedGoogle Scholar
  38. StreeckRE, MoritzKB, BeerK (1982) Chromatin diminution in Ascaris suum: nucleotide sequence of the eliminated satellite DNA. Nucleic Acids Res 10: 3495–3502PubMedGoogle Scholar
  39. SturmKS, TaylorJH (1981) Distribution of 5-methylcytosine in the DNA of somatic and germ line cells from bovine tissues. Nucleic Acids Res 9: 4537–4546PubMedGoogle Scholar
  40. SumnerAT (1972) A simple technique for demonstrating centromeric heterochromatin. Exp Cell Res 75: 304–306PubMedGoogle Scholar
  41. TakahashiE, HoriT, LawrenceJB, McNeilJ, SingerRH, O'ConnellP, LeppertM, WhiteR (1989) Human type II collagen gene (COL2A1) assigned to chromosome 12q13.1-q13.2 by in situ hybridization with biotinylated DNA probe. Jpn J Hum Genet 34: 307–311Google Scholar
  42. TaubF, ThompsonEB (1982) An improved method for preparing large arrays of bacterial colonies containing plasmids for hybridization: In site purification and stable binding of DNA on paper filters. Anal Biochem 126: 222–230PubMedGoogle Scholar
  43. ToblerH (1986) The differentiation of germ and somatic cell lines in nematodes. In: HennigW (ed) Germ line-soma differentiation. (Results and problems in cell differentiation 13) Springer, Berlin Heidelberg, pp 1–69Google Scholar
  44. ToblerH, MüllerF, BackE, AebyP (1985) Germ line-soma differentiation in Ascaris: a molecular approach. Experientia 41: 1311–1319Google Scholar
  45. ToneM, NakanoN, TakaoE, NarisawaS, MizunoS (1982) Demonstration of W chromosome-specific repetitive DNA sequences in the domestic fowl, Gallus g. domesticus. Chromosoma 86: 551–569PubMedGoogle Scholar
  46. VarleyJM, MacgregorHC, ErbaHP (1980) Satellite DNA is transcribed on lampburush chromosomes. Nature 283: 686–688PubMedGoogle Scholar
  47. VieiraJ, MessingJ (1987) Production of single-stranded plasmid DNA. Methods Enzymol 153: 3–11CrossRefPubMedGoogle Scholar
  48. WalkerPMB (1971) “Repetitive” DNA in higher organisms. Prog Biophys Mol Biol 23: 145–190CrossRefPubMedGoogle Scholar
  49. WuZ, MurphyC, GallJG (1986) A transcribed satellite DNA from the bullfrog Rana catesbeiana. Chromosoma 93: 291–297PubMedGoogle Scholar

Copyright information

© Springer-Verlag 1993

Authors and Affiliations

  • Souichirou Kubota
    • 1
  • Masaki Kuro-o
    • 1
  • Shigeki Mizuno
    • 2
  • Sei-ichi Kohno
    • 1
  1. 1.Department of Biology, Faculty of ScienceToho UniversityFunabashi, ChibaJapan
  2. 2.Laboratory of Molecular Biology, Department of Applied Biological Chemistry, Faculty of AgricultureTohoku UniversitySendai, MiyagiJapan

Personalised recommendations