Factores relacionados a los hábitos de higiene

Firstly, the results of a pilot study of Expressed Sequence Tag analysis for Chlamydomonas

were presented. A robust and routine method was developed for the isolation of ESTs from

Chlamydomonas by PCR and sixteen cDNAs chosen at random from a Chlamydomonas

cDNA library were sequenced from both ends. These sequences represent Chlamydomonas

ESTs, and were analysed for sequence similarities to other organisms and for functional motifs. Of the sixteen ESTs, two clones were shown to have exact sequence homology with well characterised nuclear genes encoding ferredoxin and ribulose-5'-bisphosphate carboxylase. A further four clones were identified that showed similarity to previously characterised Chlamydomonas genes and were putatively identified as genes encoding new members of Chlamydomonas multigene families e.g. cabll (see chapter 3). Three of the remaining clones encoded products with some significant homology to proteins from other organisms such as cytochrome p450 and a negatively phytochrome regulated protein, whilst the remaining clones failed to produce a significant match with any known protein. Whilst these latter two classes clearly represent new Chlamydomonas genes, it is impossible to confidently assign a function to them. Prehminary EST analysis of a further 84 clones produced by the EST PCR protocol further demonstrated the effectiveness of the procedure and produced some interesting results. In total, 100 clones were sequenced and were found to include copies of previously characterised Chlamydomonas genes RBCS2 and cabll, as well as the genes encoding Chlamydomonas ferredoxin and an ADP-ribosylation factor (Mamon et al. 1995). The BLAST searches also indicated the presence of number of other interesting clones in the hbrary including one encoding a soluble starch synthase. In addition, several clones encoding a product that is remarkably similar to PsaL or subunit V of photosystem I, fi"om a number of organisms. PsaL has not so far been cloned from

Chlamydomonas. Also included were a ribosomal 60S protein that showed high homology 169

to the 60S ribosomal protein fiom Arabidopsis and yeast, and another 60S acidic ribosomal protein that showed similarity to the 60S acidic ribosomal protein P2 from Trypanosoma cruzi.

6.1.2 The use of green fluorescent protein as a rep o rter fo r C hlam ydom onas

The results of the investigation of the use of Green Fluorescent Protein as a reporter molecule for Chlamydomonas have proved disappointing. It does not appear to be possible to routinely express gfp in Chlamydomonas, a problem that appears to be intrinsic to the codon usage of the alga. The DNA does not appear to be transcribed or translated, although the lack of evidence for a gfp transcript could be due to the fact that the mRNA is recognised as foreign to Chlamydomonas, and as such is rapidly degraded. It seems that the major problem for the expression of the A. victoria gene in Chlamydomonas is one of codon usage. If this is indeed the case, extensive changes to the codon usage by construction of a synthetic gene will be required if gfp is to be expressed in the alga. Such an approach has been tried but was not found to be successful [Peter Hegemann-personal communication]. This is most probably due to the fact that changing the codon usage of a gene is an unknown quantity in terms of introduction of secondary structure.

Furthermore such an approach is expensive and time consuming. With the continued emergence of an ever increasing range of mutant versions of GFP it is possible that a gfp

variant will eventually emerge that is suitable for expression in the alga. It seemed that the GFP variant EGFP might provide such an alternative, however there appears to be a further problem with expression of this variant. Lack of expression of EGFP could perhaps be due to the existence of a cryptic splice site within the coding sequence of EGFP. Investigation into the mechanism of intron splicing in Chlamydomonas has not lead to the resolution of a clear consensus splice sequence. Identification of such an element within the coding regions of a transgene may need to wait until better knowledge is available of the factors regulating RNA splicing in the alga.

6.1.3 Gene targeting and antisense dow n-regulation of nuclear genes in

C hlam ydom onas

An attempt was 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. Two pathways were investigated in which substitution of a substrate for its fluoro-analogue causes cell death in Chlamydomonas; that for UMP biosynthesis, and that for acetate utilisation. Mutants resistant to 5-FOA were created by UV mutagenesis but were subsequently found to be defective in some aspect of uracil utilisation other than the gene encoding OMPD. Furthermore, attempts to clone wild-type ompd from Chlamydomonas

were not successful and the approach was subsequently halted. Mutants affected in acetate utilisation were also produced, this time by insertional mutagenesis, which provides a tag on the affected gene. Unfortunately, due to time constraints it was not possible to fully characterise the mutants and clone the affected gene by virtue of the fact that it is tagged with an insertional mutagen. On the whole, however, this insertional mutagenesis approach has produced some encouraging results. Clearly, fluoro-analogue resistance is a promising approach for the isolation of mutants affected in some aspect of substrate metabolism. There are, of course, many alternative fluoro-analogues of metabolic substrates that could be used to isolate a Chlamydomonas mutant defective in a single enzyme of a metabolic pathway, and cloning of corresponding gene should be relatively straightforward if the mutant is produced by insertional mutagenesis. The main disadvantage of this approach is that complementation of a mutant is not always desirable as a means of selection. In order to use this method of selection with a given mutant strain, it is first necessary to cross the strain to the fluoro-analogue resistant mutant background. Also, this means of negative selection is leaky - there will be a significant background of non-homologous transformants in which the negative selectable marker has failed to integrate, has been subject to rearrangements or mutations during integration, or is poorly expressed in the transformant. However, such a system could still provide a useful means for enriching for homologous recombination events.

Finally the use of antisense expression was investigated. An antisense section of the

Chlamydomonas nuclear gene encoding oeel was introduced into the nuclear genome under the control of a strong Chlamydomonas promoter and the resulting transformants analysed for phenotypic effects and for the presence of an antisense transcript^ No effect on phenotype could be observed, and an antisense transcript could not be detected in the transformants. This leads to speculation that the lack of antisense inhibition in

Chlamydomonas could, like gfp expression, be due to a failure in translation ability. The antisense transcript is recognised as foreign to the organism and as such rapidly turned over by the cell. One prospect for antisense expression in Chlamydomonas could be to use small mRNAs which will be less susceptible to degradation due to the fact that RNase binding to its target RNA will not be possible, or to use an antisense RNA targeted to the ribosome binding site around the ATG start codon.

6 . 2 Prospects for Chlamydomonas molecular techniques