Stevens, David Roy; (1996) Nuclear Transformation and Gene Expression in Chlamydomonas reinhardtii. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Although nuclear transformation of C. reinhardtii using homologous markers for mutant rescue is now a routine procedure, attempts to express foreign genes in this alga have been conspicuously unsuccessful. Several reasons have been proposed including a biased codon usage in C. reinhardtii nuclear genes, the methylation of foreign DNA or the incompatibility/absence of untranslated elements in the introduced DNA such as promoters, polyadenylation signals and introns. In this thesis evidence is presented for the expression of a bacterial gene in C. reinhardtii. A construct in which the phleomycin resistance gene (ble) of Streptoalloteichus hindustanus is flanked by the 5' and 3' untranslated regions of a C. reinhardtii nuclear gene, was introduced into an arg7 mutant by co-transformation with the ARG7 marker. Screening of arg+ transformants revealed that ~3% are resistant to Pm. However, Southern analysis showed that ~90% of the transformant genomes contain the ble gene. Western analysis of cotransformants using antibodies against the Sh.ble protein revealed that it is present in the PmR cells. Crosses between Pm65 (a PmR transformant containing a single copy of the ble gene) and wild type cells demonstrated that the PmR phenotype segregates in predicted Mendelian ratios in progeny. Southern analysis of these progeny has revealed that the ble gene co-segregates with the Pmr phenotype. By modification of the transformation method a protocol has been developed that allows the direct selection of nuclear transformants at a low level using the ble construct. Studies into parameters affecting the rate of transformation were conducted and the results presented. The sequence of a cDNA clone for the gene RPL41 encoding the ribosomal protein L41 subunit gene is presented. The potential of using a mutated version of RPL4l as a selectable marker in nuclear transformation to confer resistance to the translation inhibitor cycloheximide is considered.

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