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Current Genetics

, Volume 26, Issue 1, pp 87–90 | Cite as

A versatile shuttle cosmid vector for the efficient construction of genomic libraries and for the cloning of fungal genes

  • Heinz D. Osiewacz
Short Communications

Abstract

A shuttle cosmid vector, pANsCos1, has been constructed for Escherichia coli and filamentous fungi. This vector contains two cos sequences separated by a single XbaI restriction site. pANsCos1 allows the efficient construction of representative genomic libraries from as little as 15–20 μg of genomic DNA. Due to the presence of a functional hygromycin B phosphotransferase gene (hph) transformation of fungal protoplasts with pANsCos1, or derivatives of it, results in the formation of hygromycin B-resistant transformants. The T7 and T3 RNA polymerase promoter sequences flanking the cloning site, in combination with two adjacent NotI sites facilitate genomic walking and the rapid construction of restriction maps of cloned inserts.

Key words

Podospora anserina Shuttle cosmid Genomic libraries Fungal transformation Genbmic walking 

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References

  1. Krumlauf R, Marzluff GA (1980) Genome organization and characterization of the repetitive and inverted repeat DNA sequences in Neurosopora crassa. J Biol Chem 255:1138–1145Google Scholar
  2. Osiewacz HD, Weber A (1989) DNA-mediated transformation of the filamentous fungus Curvularia lunata using a dominant selectable marker. Appl Microbiol Biotechnol 30:375–380Google Scholar
  3. Osiewacz HD, Clairmont A, Huth M (1990) Electrophoretic karyotype of the ascomycete Podospora anserina. Curr Genet 18: 481–483Google Scholar
  4. Osiewacz HD, Skaletz A, Esser K (1991) Integrative transformation of the ascomycete Podospora anserina: identification of the mating-type locus on chromosome VII of electrophoretically-separated chromosomes. Appl Microbiol Biotechnol 35:38–45Google Scholar
  5. Punt PJ, Oliver RJ, Dingemansee MA, Pouwels PH, van den Hondel CAMJJ (1987) Transformation of Aspergillus based on the hygromycin B resistance marker from Escherichia coli. Gene 56: 117–124Google Scholar
  6. Ridder R, Osiewacz HD (1992) Sequence analysis of the gene coding for glyceraldehyde-3-phosphate dehydrogenase (gpd) of Podospora anserina: use of homologous regulatory sequences to improve transformation efficiency Curr Genet 21:207–213Google Scholar
  7. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. Cold Spring Harbor Laboratory, Cold Spring Harbor, New YorkGoogle Scholar
  8. Turcq B, Denayrolles M, Begueret J (1990) Isolation of the two allelic incompatibility genes s and S of the fungus Podospora anserina. Curr Genet 17:297–303Google Scholar
  9. Verbeet MP, Klootwijk J, van Heerikhuizen H, Fontijn R, Vreugdenhill E, Planta RJ (1993) Molecular cloning of the rDNA of Saccharomyces rosei and comparison of its transcription region with that of Saccharomyces carlsbergensis. Gene 23:53–63Google Scholar
  10. Vollmer SJ, Yanofsky C (1986) Efficient cloning of genes of Neurospora crassa. Proc Natl Acad Sci USA 83:4869–4873Google Scholar
  11. Yelton MM, Timberlake WE, van den Hondel CAMJJ (1985) A cosmid for selecting genes by complementation in Aspergillus nidulans: selection of the developmentally-regulated yA locus. Proc Natl Acad Sci USA 82:834–838Google Scholar

Copyright information

© Springer-Verlag 1994

Authors and Affiliations

  • Heinz D. Osiewacz
    • 1
  1. 1.Angewandte Tumorvirologie, Abteilung: Molekularbiologie der AlterungsprozesseDeutsches KrebsforschungszentrumHeidelbergGermany

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