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Gene. 2001 Oct 17;277(1-2):221-9.

A Streptomyces rimosus aphVIII gene coding for a new type phosphotransferase provides stable antibiotic resistance to Chlamydomonas reinhardtii.

Author information

1
Biological Institute, St. Petersburg State University, Oranienbaumskoye sch., 2, St. Petersburg, 198904 Russia. irina.sizova@is6522.spb.edu

Abstract

Although Chlamydomonas reinhardtii serves as the most popular algal model system, no efficient enzymatic selection marker for the nuclear transformation of wild-type cells is available. We sequenced an aminoglycoside 3'-phosphotransferase gene (aph) from Streptomyces rimosus. Though the derived protein sequence is homologous to members of APH type V, it constitutes a new type, named APHVIII. Since the aphVIII gene has a codon bias similar to that of the nuclear genome of green algae, the aphVIII coding sequence was fused to the 5'- and 3'-untranslated regions of the C. reinhardtii rbcS2 gene. C. reinhardtii transformants were capable of inactivating the antibiotics paromomycin, kanamycin, and neomycin, to which wild-type cells are sensitive. After addition of the 5'-region of hsp70A as a second promoter and insertion of the rbcS2 intron I, the transformation rate increased to two transformants per 1 x 10(5) cells, which is close to the efficiency of transforming auxotrophic strains with the homologous marker arg7. Transformation with the promoter-less aphVIII led to random gene fusion at high frequency. In an aphVIII-based reporter gene assay we have found a so far unknown promoter activity of the 3'-untranslated region of rbcS2, that may promote antisense RNA synthesis from the rbcS2 gene in vivo. We conclude that the aphVIII gene is a useful marker for nuclear transformation and promoter tagging of C. reinhardtii wild-type and probably other green algae.

PMID:
11602359
[Indexed for MEDLINE]

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