REQUISITOS FÍSICOS Y QUÍMICOS DEL CEMENTO

Gene targeting using linear double stranded DNA fragments in WT E. coli is generally inefficient due to the exonucleic activity o f RecBCD. Although RecA carries out the fundamental pairing steps of recombination, other proteins are involved in other steps o f the process. Paramount is the RecBC enzyme (encoded by recB and recC). This enzyme is important in DNA unwinding, its nuclease activities providing DNA with a free end, possibly allowing RecA to bind and begin recombination. Because o f this exonucleic activity, recombination proficient strains in which the exonucleic activity o f RecBCD is inactivated are used (e.g. Russell et a l, 1989; Shevell et a l, 1988). However, mutation o f either recB or recC reduces recombination by approximately 100-fold, and this affects DNA repair. More recently an approach has been developed to overcome this difficulty by introducing Chi sequences (5'-GCTGGTGG-3') at both ends flanking the insertion (Dabert and Smith, 1997). These sequences attenuate RecBCD exonuclease activities and stimulate its recombination activity (Dixon and Kowalczykowski, 1993). However, in 1999, Karoui et a l published another method describing how gene replacement could be achieved in many WT E. coli strains following electroporation o f linear DNA. For chromosomal replacement they targeted the E. coli histidine {his) operon. A 3kb fragment o f this operon was disrupted by a kanamycin resistance gene with or without Chi sites. Following linearization and purification, WT his^ E. coli was transformed by electroporation. Kanamycin resistant colonies were patched on fresh LB-Km plates and replicated onto minimal medium lacking histidine as well as onto LB-Amp (ampicillin resistance being carried by the vector). Gene replacement events were scored as Km% His', Amp®. They found approximately 60 gene replacement events/pg o f linear DNA. However, the presence o f Chi sites on the linear DNA had no effect on gene replacement. The presence of Chi sites only stimulated recombination if E, coli cells were made competent with CaCb (Dabert and Smith, 1997). The success of recombination following electroporation suggested that this might partially inactivate RecBCD exonuclease activity, reducing degradation o f linear DNA upon its entry to the cell, while still allowing the gene replacement event to occur.

Another method to stimulate gene replacement is to use 1 recombination functions. This method is extremely efficient (Murphy, 1998). However, it requires

the use o f particular E. coli strains, limiting its range whereas the method described by Karoui et a l (1999) is compatible with many E. coli strains.

1.9.2 Gene replacem ent using p K 0 3

Hamilton et a l (1989) first described a method for gene replacement in WT E. coli using homologous recombination between the bacterial chromosome and a plasmid whose replication activity was temperature sensitive. At the non-permissive temperature, antibiotic resistance could only be maintained if integration o f the plasmid into the chromosome had occurred by homologous recombination between the cloned fragment and the bacterial chromosome. Subsequent switching o f integrant clones to the permissive temperature allowed excision o f the integrated plasmid. Depending upon the position of the second recombinational event resulting in excision, two genotypes were observed. The first retained the wild-type sequence with excision o f the plasmid containing the altered sequence. The second resulted in replacement o f the wild-type sequence with the altered sequence. There was however one major drawback: there was no selection for the loss o f vector sequence.

The method described by Hamilton et a l (1989) was first modified by Bloomfield et a l (1991) to include a counter selectable marker in the chromosome, which would facilitate allelic exchange. They used the sacB gene from B, subtilis which encodes levansucrase, an enzyme that catalyses the hydrolysis o f sucrose and levan elongation (Dedonder, 1966). When expressed in E. coli growing on media with sucrose, the sacB gene is lethal (Gay et a l, 1985). Link et a l (1997) decided to go one step further and combine the temperature sensitive origin o f replication {repA derived fi*om pSClOl) with the negative selection marker {sacB) into one replacement vector, pK 03. They also included the cat gene (encoding resistance to chloramphenicol) on the vector as a positive marker for integration and distinguishing cells harbouring vector sequences following excision. Using this vector they introduced precise insertions and deletions, as well as insertion o f a kanamycin resistance gene into both essential and non-essential E. coli genes.

1.9.2.1 Non-essential gene knockout (Link et a l, 1997)

The gene, yjb, encoding a highly abundant protein, o f unknown function was disrupted using a kanamycin resistance gene and cloned into pK 03. Following transformation of a recombinant proficient E. coli strain at the permissive

temperature, 30°C, integrants were selected at the non-permissive temperature (43°C) by kanamycin and chloramphenicol resistance. Selected integrants were then serially diluted, at the permissive temperature onto plates supplemented with 5% sucrose and kanamycin. Viable colonies were classed as “potential knockouts” as kanamycin selected for the disrupted gene and growth on sucrose indicated excision and loss of pK03. To confirm the loss o f vector sequence, the potential positives were replica plated onto agar supplemented with 5% sucrose and chloramphenicol. O f these colonies, 98% were chloramphenicol sensitive. This indicated the loss o f plasmid sequences and gene replacement, which was confirmed by PCR.

1.9.2.2 Essential gene knockout (Link et a l, 1997)

Two known essential genes pepM (a map gene encoding methionine aminopeptidase) and rpsB (a ribosomal protein encoding gene) were disrupted with a kanamycin resistance gene and cloned into pK03. Following transformation at the permissive temperature, integration o f pK 03 containing the disrupted gene was selected at the non-permissive temperature as above. Again potential integrants were serially diluted onto plates supplemented with 5% sucrose and kanamycin at 30°C. The positives were then replica plated onto 5% sucrose and chloramphenicol. This time all o f the kanamycin resistant colonies remained chloramphenicol resistant. This indicated that plasmid sequences were still present in the cells and PCR confirmed that no knockout had occurred. Instead, excision o f pK 03 containing the disrupted gene had occurred. Any colonies containing pK 03, with the disrupted gene, thereby allowing selection on both kanamycin and chloramphenicol in the presence of 5% sucrose, were attributed to either a mutation in sacB or a compensatory mutation in the genome rendering the expression o f sacB non-lethal.

As will be described later, I chose this method to determine if disruption of WT E. coli at theyc/’24 locus followed the pattern described by Link et a l (1997) for an essential or for an non-essential gene.

1.9 Sum m ary

The discovery o f the malarial plastid genome was unexpected and the source organism that contained the plastid o f apicomplexans remains to be determined. Although descent from a dinoflagellate/ciliate clade was implied from earlier

phylogenetic studies o f nucleus encoded rRNA genes, ciliates do not contain plastids. Unfortunately the high evolutionary rate o f the plDNA has made it difficult to reach any clear conclusions on a red or green algal origin and this point is still debated. What seems clear however, is that the plastid originated by secondary endosymbiosis and the highly conserved gene content, and the order o f gene sequences suggests the apicoplast evolved from a single photosynthetic progenitor. The high level of conservation, as well as evidence for transcription, translation, sensitivity to antibiotics and protein import all indicate a functional role for the apicoplast. What this is remains to be determined.

The plastid is o f particular interest as a potential drug target and although research in this field is in its early stages, it aims to provide treatments effective against one of the most devastating diseases o f mankind.

It is hoped that due to the plastid’s prokaryotic nature, drugs affecting plastid biosynthetic pathways which do not affect those o f eukaryotes may be developed. Initial evidence, although mainly in vitro, suggests that antibiotics can be targeted to the plastid resulting in inhibition o f parasite growth. In addition to inhibition of organellar protein synthesis, drugs may also be developed which affect protein import into the plastid. This might involve the use of herbicides known specifically to inhibit the non-photosynthetic pathways of plastids. My thesis is concerned with establishing some indication o f the function o f the large open reading frame ORF 470 {ycf 24) encoded on the plastid genome. If this gene is essential even Y cf 24 may become a potential drug target.

Aims

I. This study aimed to “knock-out” ycflA in bacterial surrogate systems thereby gaining insight into the function o f the gene in Plasmodium falciparum. My first approach was gene inactivation in the cyanobacterium, Synechocystis PCC6803, replacing some copies of the multi-copy wild type gene with y t / 24 disrupted with either a kanamycin or a streptomycin/spectinomycin resistance cassette. Following transformation, recombinants were selected and their growth characteristics (phenotype) analysed with a view to determining the underlying effect o f gene disruption. Similar experiments were planned using the E. coli single copy orthologue o ïy c flA .

II. A second objective was to over-express E. coli y c f 24. As well as phenotypic observations, successful over-expression would allow purification o f the protein for antibody production, which could give information on the location o f the y c f 24 gene product and perhaps it’s cellular function.

Chapter 2. Materials and methods