High copy number integration into the ribosomal DNA of the yeast Phaffia rhodozyma
| Autor: | D.A.M. Gutker, A.J.J. van Ooyen, A.C.H.M. Renniers, Jan C. Verdoes, Jan Wery |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 1997 |
| Předmět: |
Genetic Markers
Transposable element Genes Fungal Drug Resistance Gene Dosage Biology DNA Ribosomal Transformation Industrial Microbiology Transformation Genetic Plasmid Mitotic stability Kanamycin Genetics Industriële microbiologie Cloning Molecular Promoter Regions Genetic Gene Ribosomal DNA Genomic organization VLAG Electrophoresis Agar Gel Homologous integration Astaxanthin Fungal genetics General Medicine Geneticin Molecular biology Actins Electrophoresis Gel Pulsed-Field Blotting Southern Transformation (genetics) Gentamicins Ploidy DNA Probes Plasmids |
| Zdroj: | Gene 184 (1997) Gene, 184, 89-97 |
| ISSN: | 0378-1119 |
| Popis: | This report describes a transformation system leading to stable high copy number integration into the ribosomal DNA (rDNA) of the astaxanthin-producing yeast Phaffia rhodozyma. A plasmid was constructed that contains the transposon Tn5 encoded kanamycin resistance gene (KmR) fused in frame to the 5'-terminal portion of the Phaffia actin gene. This marker, driven by the Phaffia actin promoter, confers resistance to G418 (Geneticin). The plasmid also contains a rDNA portion that comprises the 18S rDNA and promotes high copy integration leading to stable Phaffia transformants that maintained the plasmid at high copy number after 15 generations of non-selective growth. Phaffia, strain CBS 6938, was found to contain the rDNA units in clusters distributed over three chromosomes with a total copy number of 61. Phaffia transformants were shown to have over 50 copies of pGB-Ph9 integrated in tandem in chromosomes that contain rDNA loci. The chromosomal shifts that occur as a result of these integrations as shown by pulsed field electrophoresis strongly suggest that Phaffia is haploid. |
| Databáze: | OpenAIRE |
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