Stable nuclear transformation of Chlamydomonas using the Chlamydomonas gene for nitrate reductase.

Abstract

Abstract. We have developed a nuclear transformation system for Chlamydomonas reinhardtii, using microprojectile bombardment to introduce the gene encoding nitrate reductase into a nitl mutant strain which lacks nitrate reductase activity. By using either supercoiled or linear plasmid DNA, transformants were recovered consistently at a low efficiency, on the order of 15 transformants per microgram of plasmid DNA. In all cases the transforming DNA was integrated into the nuclear genome, usually in multiple copies. Most of the introduced copies were genetically linked to each other, and they were unlinked to the original nit1 locus. The transforming DNA and nit + phenotype were stable through mitosis and meiosis, even in the absence of selection, nit1 transcripts of various sizes were expressed at levels equal to or greater than those in wild-type nit + strains. In most transformants, nitrate reductase enzyme activity was expressed at approximately wild-type levels. In all transformants, nit1 mRNA and nitrate reductase enzyme activity were repressed in cells grown on ammonium medium, showing that expression of the integrated nit1 genes was regulated normally. When a second plasmid with a nonselectable gene was bombarded into the cells along with the nit1 gene, transformants carrying DNA from both plasmids were recovered. In some cases, expression of the unselected gene could be detected.With the advent of nuclear transformation in Chlamydomonas, it becomes the first photosynthetic organism in which both the nuclear and chloroplast compartments can be transformed.

Oda5p, a Novel Axonemal Protein Required for Assembly of the Outer Dynein Arm and an Associated Adenylate Kinase

“…All transformations were done using the glass bead method as described previously (Kindle et al, 1989;Koutoulis et al, 1997). The insertional mutant 45BO3 was generated by transforming CC-2454 cells with plasmid pMN24 containing the Chlamydomonas NIT1 gene.…”

Transient Gene Expression System Established in Porphyra yezoensis Is Widely Applicable in Bangiophycean Algae

“…Stable transformation systems have already been established in microalgae (Hallmann 2007), such as volvocine green algae, unicellular Chlamydomonas reinhardtii (Debuchy et al 1989;Kindle et al 1989), Dunaliella salina (Geng et al 2003(Geng et al , 2004) and multicellular Volvox carteri (Schiedlmeier et al 1994), all of which have long been favorite experimental organisms for genetic and molecular biological studies. Recently, eukaryotic green alga Ostreococcus tauri, which is an attractive microalgal model because of its minimal cellular organization and compact genome, has also become transformable (Corellou et al 2009;Moulager et al, 2010;Heidje et al, 2010).…”

Flagellar radial spoke: A model molecular genetic system for studying organelle assembly

“…The ease of isolating such motility mutants led to the preeminence of Chlamydomonas as a model system for studies of flagellar biology [reviewed by Luck, 1984;Huang, 19861. The genes for many flagellar proteins have recently been cloned [Schloss et al, 1984;Williams et al, 1986Williams et al, , 1989Mitchell, 1989;Mitchell and Kang, 19911, and methods have been developed to transform the Chlamydomonas nuclear genome [Kindle et al, 1989;Kindle, 1990;Diener et al, 19901; thus, a full range of genetic and reverse genetic tools are available in this organism to dissect the roles of individual components in flagellar motility.…”