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Journal List > Nucleic Acids Res > v.21(3); Feb 11, 1993

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Nucleic Acids Res. 1993 February 11; 21(3): 497–504.
PMCID: PMC309145
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Isolation of telomeric DNA from the filamentous fungus Podospora anserina and construction of a self-replicating linear plasmid showing high transformation frequency.
J P Javerzat, V Bhattacherjee, and C Barreau
Laboratoire de Génétique, UA CNRS 542, Université de Bordeaux II, Talence, France.
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Abstract
It has been previously shown that linear plasmids bearing Tetrahymena telomeric sequences are able to replicate autonomously in the filamentous fungus Podospora anserina (1). However, autonomous replication occurs in only 50-70% of the transformants, suggesting a defect in the recognition of the Tetrahymena telomeric template by the putative P. anserina telomerase so that only a fraction of entering DNA is stabilized into linear extrachromosomal molecules. We have cloned DNA sequences added to the Tetrahymena (T2G4)n ends of the linear plasmid. Nucleotide sequencing showed that these sequences are exclusively composed of T2AG3 repeat units. Hybridization experiments of Bal31 treated DNA showed that T2AG3 repeats are confined within 200 bp in chromosomal P. anserina telomeres. A new plasmid has been constructed so that after linearization, the terminal sequences contain T2AG3 repeats. This linear molecule transforms P. anserina with a high frequency (up to 1.75 x 10(4) transformants/micrograms), autonomous replication occurs in 100% of the transformants and the plasmid copy number is about 2-3 per nucleus. These results underscore the importance of the telomeric repeat nucleotide sequence for efficient recognition as functional telomeric DNA in vivo and provide the first step toward the development of an artificial chromosome cloning system for filamentous fungi.
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Selected References
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  • Perrot M, Barreau C, Bégueret J. Nonintegrative transformation in the filamentous fungus Podospora anserina: stabilization of a linear vector by the chromosomal ends of Tetrahymena thermophila. Mol Cell Biol. 1987 May;7(5):1725–1730. [PubMed]
  • Blackburn EH. The molecular structure of centromeres and telomeres. Annu Rev Biochem. 1984;53:163–194. [PubMed]
  • Blackburn EH. Structure and function of telomeres. Nature. 1991 Apr 18;350(6319):569–573. [PubMed]
  • Blackburn EH, Gall JG. A tandemly repeated sequence at the termini of the extrachromosomal ribosomal RNA genes in Tetrahymena. J Mol Biol. 1978 Mar 25;120(1):33–53. [PubMed]
  • Klobutcher LA, Swanton MT, Donini P, Prescott DM. All gene-sized DNA molecules in four species of hypotrichs have the same terminal sequence and an unusual 3' terminus. Proc Natl Acad Sci U S A. 1981 May;78(5):3015–3019. [PubMed]
  • Shampay J, Szostak JW, Blackburn EH. DNA sequences of telomeres maintained in yeast. Nature. 1984 Jul 12;310(5973):154–157. [PubMed]
  • Emery HS, Weiner AM. An irregular satellite sequence is found at the termini of the linear extrachromosomal rDNA in Dictyostelium discoideum. Cell. 1981 Nov;26(3 Pt 1):411–419. [PubMed]
  • Greider CW, Blackburn EH. Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell. 1985 Dec;43(2 Pt 1):405–413. [PubMed]
  • Greider CW, Blackburn EH. The telomere terminal transferase of Tetrahymena is a ribonucleoprotein enzyme with two kinds of primer specificity. Cell. 1987 Dec 24;51(6):887–898. [PubMed]
  • Greider CW, Blackburn EH. A telomeric sequence in the RNA of Tetrahymena telomerase required for telomere repeat synthesis. Nature. 1989 Jan 26;337(6205):331–337. [PubMed]
  • Morin GB. The human telomere terminal transferase enzyme is a ribonucleoprotein that synthesizes TTAGGG repeats. Cell. 1989 Nov 3;59(3):521–529. [PubMed]
  • Shippen-Lentz D, Blackburn EH. Functional evidence for an RNA template in telomerase. Science. 1990 Feb 2;247(4942):546–552. [PubMed]
  • Pluta AF, Dani GM, Spear BB, Zakian VA. Elaboration of telomeres in yeast: recognition and modification of termini from Oxytricha macronuclear DNA. Proc Natl Acad Sci U S A. 1984 Mar;81(5):1475–1479. [PubMed]
  • Hahnenberger KM, Baum MP, Polizzi CM, Carbon J, Clarke L. Construction of functional artificial minichromosomes in the fission yeast Schizosaccharomyces pombe. Proc Natl Acad Sci U S A. 1989 Jan;86(2):577–581. [PubMed]
  • Cross SH, Allshire RC, McKay SJ, McGill NI, Cooke HJ. Cloning of human telomeres by complementation in yeast. Nature. 1989 Apr 27;338(6218):771–774. [PubMed]
  • Brown WR. Molecular cloning of human telomeres in yeast. Nature. 1989 Apr 27;338(6218):774–776. [PubMed]
  • Razanamparany V, Bégueret J. Positive screening and transformation of ura5 mutants in the fungus Podospora anserina: characterization of the transformants. Curr Genet. 1986;10(11):811–817. [PubMed]
  • Turcq B, Bégueret J. The ura5 gene of the filamentous fungus Podospora anserina: nucleotide sequence and expression in transformed strains. Gene. 1987;53(2-3):201–209. [PubMed]
  • Burke DT, Carle GF, Olson MV. Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors. Science. 1987 May 15;236(4803):806–812. [PubMed]
  • Marchuk D, Collins FS. pYAC-RC, a yeast artificial chromosome vector for cloning DNA cut with infrequently cutting restriction endonucleases. Nucleic Acids Res. 1988 Aug 11;16(15):7743–7743. [PubMed]
  • Cross S, Lindsey J, Fantes J, McKay S, McGill N, Cooke H. The structure of a subterminal repeated sequence present on many human chromosomes. Nucleic Acids Res. 1990 Nov 25;18(22):6649–6657. [PubMed]
  • Vieira J, Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. [PubMed]
  • Oka A, Sugisaki H, Takanami M. Nucleotide sequence of the kanamycin resistance transposon Tn903. J Mol Biol. 1981 Apr 5;147(2):217–226. [PubMed]
  • DuBridge RB, Tang P, Hsia HC, Leong PM, Miller JH, Calos MP. Analysis of mutation in human cells by using an Epstein-Barr virus shuttle system. Mol Cell Biol. 1987 Jan;7(1):379–387. [PubMed]
  • Bégueret J, Razanamparany V, Perrot M, Barreau C. Cloning gene ura5 for the orotidylic acid pyrophosphorylase of the filamentous fungus Podospora anserina: transformation of protoplasts. Gene. 1984 Dec;32(3):487–492. [PubMed]
  • Bergès T, Barreau C. Heat shock at an elevated temperature improves transformation efficiency of protoplasts from Podospora anserina. J Gen Microbiol. 1989 Mar;135(3):601–604. [PubMed]
  • Wang SS, Zakian VA. Sequencing of Saccharomyces telomeres cloned using T4 DNA polymerase reveals two domains. Mol Cell Biol. 1990 Aug;10(8):4415–4419. [PubMed]
  • Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. [PubMed]
  • Szostak JW, Blackburn EH. Cloning yeast telomeres on linear plasmid vectors. Cell. 1982 May;29(1):245–255. [PubMed]
  • Farr C, Fantes J, Goodfellow P, Cooke H. Functional reintroduction of human telomeres into mammalian cells. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):7006–7010. [PubMed]
  • Horowitz H, Haber JE. Identification of autonomously replicating circular subtelomeric Y' elements in Saccharomyces cerevisiae. Mol Cell Biol. 1985 Sep;5(9):2369–2380. [PubMed]
  • Kiss GB, Amin AA, Pearlman RE. Two separate regions of the extrachromosomal ribosomal deoxyribonucleic acid of Tetrahymena thermophila enable autonomous replication of plasmids in Saccharomyces cerevisiae. Mol Cell Biol. 1981 Jun;1(6):535–543. [PubMed]
  • Powell WA, Kistler HC. In vivo rearrangement of foreign DNA by Fusarium oxysporum produces linear self-replicating plasmids. J Bacteriol. 1990 Jun;172(6):3163–3171. [PubMed]
  • de Lange T. Human telomeres are attached to the nuclear matrix. EMBO J. 1992 Feb;11(2):717–724. [PubMed]
  • Price CM. Telomere structure in Euplotes crassus: characterization of DNA-protein interactions and isolation of a telomere-binding protein. Mol Cell Biol. 1990 Jul;10(7):3421–3431. [PubMed]
  • Price CM, Cech TR. Properties of the telomeric DNA-binding protein from Oxytricha nova. Biochemistry. 1989 Jan 24;28(2):769–774. [PubMed]
  • Conrad MN, Wright JH, Wolf AJ, Zakian VA. RAP1 protein interacts with yeast telomeres in vivo: overproduction alters telomere structure and decreases chromosome stability. Cell. 1990 Nov 16;63(4):739–750. [PubMed]
  • Lustig AJ, Kurtz S, Shore D. Involvement of the silencer and UAS binding protein RAP1 in regulation of telomere length. Science. 1990 Oct 26;250(4980):549–553. [PubMed]
  • Murray AW, Szostak JW. Construction of artificial chromosomes in yeast. Nature. 1983 Sep 15;305(5931):189–193. [PubMed]
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