Western analysis of antisense transformants

As high fluorescence of the transformants was not apparent, the five positive transformants were further screened by western analysis for a decreased amount of OEEl protein using equal amounts of protein extract. Wild-type total cellular protein was used as a positive control and FuD44 total cellular protein as the negative control. The resulting western blot

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Figure 5.17 Fluorescence analysis o f oeea transformants

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Figure 5.18 Southern analysis o f poeea transformants. 10 pg of total genomic DNA extracted from the poeea transformants and from wild-type cells were digested with Pvu/7 and separated on a 1% agarose gel by electrophoresis. The DNA was transferred to a nylon filter by Southern blotting and probed with a 0.92 kb fragment o f labelled oeel cDNA DNA. The filtered was washed for 2 x 20 minutes in 0.1% SDS, 2x SSC at room temperature, then for 2x 20 minutes in 0.1% SDS, 0.2 X SSC at 65 °C. The filter was then autoradiographed fo r 24 hours at -70 °C. The figure shows the presence o f genomic oeel as two bands present at 4100 bp and 750 bp. Please note that the reason the same binding is not seen in the wild-type track is that the wild-type DNA had apparently been degraded and was seen as a smear on the agarose gel. Positive transformants show the presence o f bands at 1000 bp and 378 bp.

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Figure 5.19Western analysis of poeea transformants. Total protein extracts (measured on an equal chlorophyll basis equivalent to 10 pg o f chlorophyll) from the positive poeea transformants were loaded onto a tris-tricine gel. The same quantity o f protein extracted from FuD44, a mutant with a disrupted copy o f the wild-type oeel gene was used as a negative control, and total protein extract from wild type cells was used as a positive control. The proteins were separated by gel electrophoresis and transferred to a nitrocellulose filter. The resulting blot was probed with an antibody raised against p oeel protein from pea. Primary antibody binding was detected using a horse radish peroxidase labelled secondary antibody and subsequently autoradiography.

Note that there is no significant decrease in the amount o f OEEl protein detected relative to wild- type. Some OEE protein is also seen in the Fud44 null mutant. This could be due to ‘spill-over' during gel loading, or to the fact that reversion of the mutant population may have occurred during maintenance on agar plates.

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Figure 5.20 Northern analysis o f positive poeea transformants showing a) RNA gel probed using a 0.92 kb fragment o f labelled oee cDNA, and b) resulting autorad. Total RNA from WT and poeea transformants was separated on a 0.5% denaturing agarose gel, and transferred to a nitrocellulose membrane. Since a positive signal cannot be detected fo r the genomic oee 1 gene despite abundant amounts o f RNA present on the gel, this experiment cannot be interpreted as negative for the presence of an antisense transcript.

was probed with poly-clonal antibodies raised against pea OEEl protein (donated by Professor John Gray, University of Cambridge). The amount of OEEl protein was found to be constant in all of the transformants assayed relative to FuD44 which showed a significant decrease in the amount of OEEl protein (figure 5.19).

This evidence suggests that although the antisense DNA may be transcribed, it has no discernible effects on OEEl protein levels. It would therefore be logical to assume that antisense downregulation of the oeel gene does not take place in Chlamydomonas.

However evidence from tobacco has shown that antisense down-regulation of the oeel gene does not affect the corresponding protein levels, indicating that the mRNA levels for this component is not rate limiting for protein accumulation and that severely reduced amounts of these transcripts still allow normal plant development (Palomares, Herrmann et al. 1993). For this reason northern analysis was carried out in order to determine whether the antisense construct is transcribed in the positive transformants, and whether transcription of the antisense fragment has a discernible effect on wild-type mRNA levels.

5.2.10 N orthern analysis of antisense tran sfo rm an ts

The transformants were subject to northern analysis to assay for the presence of an antisense transcript. No antisense transcript was detected in the antisense transformants. The northern analysis also failed to detect mRNA from the single-copy nuclear gene encoding oeel,

although the positive DNA control was detected (figure 5.20). This indicates that the DNA probe is not sensitive enough to detect the presence of an antisense transcript, and the results were therefore inconclusive. Greater sensitivity could be achieved through use of a radiolabelled RNA probe, synthesised by transcription in vitro from the poeea plasmid, however time constraints meant that it was not possible to perform this experiment.

5.3 Discussion

5.3.1 Development of a negative selectable m ark e r for C hlam ydom onas

Although the strategies outlined above for the development of a negative selectable marker for Chlamydomonas have failed to yield a suitable marker gene in the short term, given a longer time scale it is possible that both strategies could yield a suitable candidate.

Presuming that Chlamydomonas does not have an alternative pathway for uracil biosynthesis that allows it to do without its UMP biosynthetic pathway, then the FOA resistant mutants isolated so far must be FOA assimilation mutants. In this case, screening of sufficient colonies should eventually yield a mutant affected in the UMP biosynthetic pathway that is both 5-FOA resistant and uracil-requiring. Isolation of wild-type

Chlamydomonas ompd may be possible using a different cDNA library, or by RACE (Rapid Amplification of cDNA Ends) from Chlamydomonas total mRNA (Frohman 1990).

The RACE protocol generates cDNA by using PCR to amplify copies of the region between a single point in the transcipt and the 3’ or 5’ end. To use the RACE protocol, one must raise a primer to a short section of the DNA sequence and a primer that anneals to the natural 5’ end or 3’ end or a synthetic poly-A tail.

The insertional mutagenesis strategy for isolation of FAM resistant mutants was successful in that the approach has been shown to yield many hundreds of transformants with the required phenotype. Analysis of these mutants by Southern analysis using lac-a DNA as a probe should show which mutants have become resistant to FAM due to insertion of lac-a DNA to produce a suitable mutant background for genomic complementation. The acetamidase utilisation pathway is complex and there are several candidate genes other than acetamidase that will yield a FAM resistant phenotype. Cloning of the wild-type gene responsible for the mutant phenotype is simplified by the fact that the mutant version of the gene should be tagged with lac-a DNA, and with a greater number of target genes for isolation of the expected phenotype this approach is more likely to provide a means of negative selection than isolation of FOA resistant mutants.