Madagan, Kathryn Louise; (1998) Tools for nuclear transformation in Chlamydomonas reinhardtii. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Due to its simple life cycle and classical Mendelian genetics the green alga Chlamydomonas reinhardtii has long been used as a model system for the study of flagellar function, photosynthesis and response to light. Transformation of the nuclear genome has now become routine and with this the development of a variety of promoters, transformation markers and methods for genomic complementation and insertional mutagenesis has taken place. However, in order to exploit fully the potential of Chlamydomonas, further procedures for molecular genetic investigation are required. For example, it is desirable to develop a detailed molecular map of the Chlamydomonas nuclear genome. Also there is a need to find a suitable reporter molecule that can act as a marker for gene expression and protein localisation. Other desirable advances would include a way of targeting specific genes by selection for rare homologous recombination events and a means for down- regulation of genes by the application of antisense technology. This thesis presents the results of investigation into possible new tools for nuclear transformation. Firstly, the results of a pilot study of analysis of Expressed Sequence Tags from Chlamydomonas are presented. Sixteen cDNAs chosen at random from a Chlamydomonas cDNA library were sequenced. The sequences were analysed for sequence similarities to other organisms and for functional motifs. Secondly, the use of Green Fluorescent Protein as a reporter molecule for Chlamydomonas was investigated. The GFP coding sequence under control of a Chlamydomonas promoter was introduced into the nuclear genome and the transformants examined for expression of Green Fluorescent Protein. Thirdly attempts were made to develop a negative selectable marker for homologous transformation based on the creation of mutants defective in uracil or acetate utilisation and their restoration to wild-type phenotype by transformation with the wild-type gene. Finally the use of antisense expression was investigated. An antisense section of the Chlamydomonas nuclear gene encoding oxygen evolving complex 1 was inserted into the genome of Chlamydomonas under the control of a strong Chlamydomonas promoter and the resulting transformants analysed for phenotypic effects and the presence of an antisense transcript.

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