EL DESDOBLAMIENTO DEL CICLO

The final decision was my choice o f vector. Only plasmid D N A can be transformed into yeast. cD N A can be cloned directly into plasmid vectors or into phagemid vectors, by which the library is initially made using a bacteriophage X vector, that is subsequently converted to plasmid form. Plasmid libraries require large amounts o f cD N A and involve the highly inefficient step o f transforming plasmids into E. coli. B y contrast, the use o f bacteriophage allows high titre libraries to be constructed from small amounts o f cD N A because the packaging o f the D N A into infectious particles is very efficient, as is the infection o f

bacteriophage into E. coli. This was my method o f choice as the use o f phagemid rather than plasmid D N A offered the best chance o f obtaining high titre libraries.

A myriad o f library yeast expression vectors are available (Elledge et al., 1993; Rose and Broach, 1991). Besides the alternatives o f plasmid or phagemid vector, they also vary in their choice o f selectable marker, yeast promoter, plasmid copy number in yeast cells and cloning strategy. It is important to use a yeast selectable marker for which the recipient strain o f yeast contains a corresponding non-reverting mutant allele. There is however some

leeway since the various triple cln strains contain common but different mutant alleles. Yeast promoters may be constitutive, such as the A D H l and GAP promoters, or

regulated such as the G A Ll promoter which is induced in the presence o f galactose (Rose and Broach, 1991). There are drawbacks to the use o f either system. Constitutive expression o f a heterologous cyclin may be toxic to the yeast cell, whilst the G A Ll promoter suffers the disadvantage that many yeast strains grow poorly on galactose and transformation o f strains grown on galactose is inefficient.

Plasmid copy number can be manipulated by the introduction o f ARS and CEN

sequences which result in a single copy per cell or by using a 2 p origin causing replication to between 25 and 100 copies per cell. There seems to be a window o f heterologous cyclin expression levels within which the cyclin defect can be overcome, but too little protein is insufficient to rescue the defect and too much expression kills the cells. The tolerable level, which is likely to vary for each individual cyclin, can be obtained by a combination o f promoter strength and plasmid copy number in the cell. In the different library vectors used

for {clnl, cln2, cln3) complementation to date, each o f the parameters discussed has varied.

If complementation is not achieved with a cD N A library cloned into one type o f vector, then it may be necessary to repeat the selection with cD N A cloned into a different vector.

I chose to use two different yeast expression vectors, the X,ADH and ^YES phagemids, designed by Steve Elledge (Elledge et a l , 1993). These two vectors differed only in the choice o f yeast promoter that they encoded. Reassuringly, libraries had already been successfully made in both vectors (Elledge et a l , 1993). Those synthesised with XYES had been used to complement S. cerevisiae mutations (Segel et a l , 1992; Elledge et a l , 1991) and other members o f my laboratory had already successfully screened libraries cloned into the X,YES vector, though not by yeast complementation (Jane Kirk, pers. comm.).

The vectors and cD N A cloning strategy that I used are illustrated in figure 4.2. The bacteriophage D N A (shown in figure 4.2A) was derived from XgX.6 and was designed for optimal growth (Elledge et a l, 1993). Figure 4.2B shows the region o f the bacteriophage D N A which encodes the pADH or pYES plasmids. This region is flanked by lox sites at which recombination occurs to produce circularised plasmid D N A when the bacteriophage are infected into a strain o f bacteria expressing the ere recombinase. This simple conversion procedure allows easy recovery o f the inserts and subsequent introduction o f the plasmid into yeast.

pADH contains the strong, constitutive alcohol dehydrogenase A D H l promoter whilst pYES contains the strong but regulated galactokinase G A Ll promoter. Since both vectors used the same cloning strategy, I could clone my final cD N A separately into both o f them and increase my chance o f complementation by screening in a background o f either

constitutive or regulated gene expression. The plasmids contain A R S l and CEN4 sequences and are thus expressed at a level o f one copy per cell. The expression directed by the strong yeast promoters should compensate, if required, for this low copy number. The plasmids contain the common URA3 selectable marker which allows selection for plasmid

maintenance.

The yeast promoter and E. coli lac promoter are located in a convergent orientation on opposite sides o f the X hol cloning site, shown in figure 4.2B. The non-directional nature o f the cloning strategy means that half o f the cD N As are expressed from each promoter. Expression o f potentially toxic genes in library construction steps involving bacteria, which could bias the library, was minimised by introducing glucose into the medium to repress the

lac promoter.

L eft X Arm Notl 19.6 kb L,__ Not! 27.4 kb J___ lox lox Plasm id coding region Right X Arm X A D W A. YES 42.6 kb

B

P 7 7 7 ^ J

1 / / / / A

lox Xhol lox

pA D H / pY ES 7.8 kb p T -F illed V ector CT G A G C T Xhol + T Adaptors C G A G W C W D ic tyos telium cD N A Adaptors W C W G A G C T -Filled V ector T C G A G W W T C W W X h o l+ T

Figure 4.2 The cDNA library cloning vectors

(A) The A.ADH / A,YES bacteriophages. A.ADH encodes the yeast A D H l promoter whilst A,YES contains the G A L l promoter. All other regions of the two bacteriophages are identical. The region between the lox sites can be converted to circularised plasmid by a site-specific recombination event.

(B) The pADH / pYES plasmids. These contain the yeast A R S l origin of replication, the CEN4 centromeric sequence, either the A D H l or G A L l promoter, the HIS3

transcriptional terminator and the URA3 selectable marker. The plasmids also encode the E. coli C olE l origin of replication, the lac promoter and the amp resistance gene. (C) The insert region of pADH / pYES. The blunt ended cDNA is ligated to 1 Im er / Smer phosphorylated adaptors. This product is in turn ligated to X hol cut, T-filled plasm id vector. The T-filling prevents self-ligation of the vector but allows ligation to the adaptor-flanked cDNA.

The library cloning strategy is shown in figure 4.2C. The vector was cut with X hol then a single deoxythymidine residue was added to partially fill in each overhanging end. The vector termini were then compatible with those o f adaptors which had been ligated onto the cD N A ends. The adaptors were short, double-stranded oligonucleotides comprised o f two strands o f unequal length. The T-filling step prevented the vector from self-ligating thus minimising the background o f empty vector in the library. It also meant that the overhanging ends o f the adaptors were not self-compatible, so that only a single adaptor could ligate onto each cD N A end. Thus, using this scheme was advantageous because the cD N A did not need to be methylated and a further restriction digest was not needed, as is the case when linkers are used (Wu et a l , 1987). The strategy also did not suffer from the usual disadvantage o f using adaptors - the requirement that one o f the oligonucleotides needs to be

unphosphorylated at the 5' end to prevent self-ligation, which reduces ligation efficiency to the cD N A. The sequence o f the adaptors used enabled the X hol sites to be recreated so that insert D N A could later easily be isolated from the vector D N A by a simple restriction digest.