An engineered Streptomyces hygroscopicus aph 7" gene mediates dominant resistance against hygromycin B in Chlamydomonas reinhardtii.
We have developed a positively selectable marker for the green alga Chlamydomonas reinhardtii using the Streptomyces hygroscopicus aminoglycoside phosphotransferase gene (aph7"). Its expression is controlled by C. reinhardtii regulatory elements, namely, the beta2-tubulin gene promoter in combination with the first intron and the 3' untranslated region of the small subunit of ribulose bisphosphate carboxylase, rbcS2. C. reinhardtii cell-wall deficient and wild-type strains were transformed at rates up to 5 x 10(-5) with two constructs, pHyg3 and pHyg4 (intron-less). Transformants selected on plates with 10 microg/ml hygromycin B exhibited diverse levels of resistance of up to 200 microg/ml that were stably maintained for at least seven months; they contained two to five copies of the construct integrated in their genomes. Transcription of the chimeric aph7" gene, correct splicing of the rbcS2 intron, and polyadenylation of the transcripts have been verified by sequencing of RT-PCR products. Average co-transformation rates using pHyg3 and a second selectable plasmid were about 11%. This advocates the hygromycin-resistance plasmid, pHyg3, as a new versatile tool for the transformation of a broad range of C. reinhardtii strains without the sustained need for using auxotrophic mutants as recipients.
Berthold
Peter
P
Lehrstuhl für Genetik, Universität Regensburg, NWFIII, Universitätsstrasse 31, D - 93040 Regensburg, Germany.
Schmitt
Rüdiger
R
Mages
Wolfgang
W
D020413:3' Untranslated Regions / Q000235:genetics; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics* / Q000502:physiology; D001483:Base Sequence; D016825:Chlamydomonas reinhardtii / Q000187:drug effects / Q000235:genetics*; D004305:Dose-Response Relationship, Drug; D004351:Drug Resistance / Q000235:genetics; D005798:Genes, Bacterial; D005813:Genes, Synthetic; D006921:Hygromycin B / Q000378:metabolism / Q000494:pharmacology*; D007438:Introns / Q000235:genetics; D019868:Kanamycin Kinase / Q000235:genetics* / Q000502:physiology; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D026723:Polyadenylation; D011401:Promoter Regions, Genetic / Q000235:genetics; D015202:Protein Engineering; D012323:RNA Processing, Post-Transcriptional; D012326:RNA Splicing; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000502:physiology; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics; D012641:Selection, Genetic; D013302:Streptomyces / Q000235:genetics*; D014158:Transcription, Genetic; D014170:Transformation, Genetic; D014404:Tubulin / Q000235:genetics
D020413:3' Untranslated Regions; D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D014404:Tubulin; D006921:Hygromycin B; D019868:Kanamycin Kinase; D012273:Ribulose-Bisphosphate Carboxylase