PROCEDIMIENTOS METODOLÓGICOS

The QIAexpressionist kit w as used to m ake a His- tagged fusion protein, according to the m anufacturers protocols. In sum m ary, a suitable fragm ent from the gene of interest w as ligated into an expression vector and transform ed into E.Coli. strain M15. These cells w ere grow n up u n d e r selection w ith kanam ycin and am picillin in LB w ith shaking and expression w as induced by addition of Im M IPTG once a required cell density had been reached. Protein was harvested from this culture at hourly intervals following induction, purified on a nickel colum n to w hich the H is-tag binds until elution by EDTA, and visualised via ID SDS-PAGE on a 12.5% gel. V arious induction conditions w ere investigated in an attem p t to m axim ise protein yield, e.g. induction at 30°C, enrichm ent of the grow th m edium w ith extra (2%) glucose, induction at different cell density and increased (200|Lig.mLi) ampicillin concentration.

2.2.S.2. One-dimensional SDS polyacrylamide gels (ID-PAGE). (Laemmli, 1970).

These gels w ere cast in the Bio-Rad M ini-Protean II apparatus, using 1mm spacers and assem bled as described by the m anufacturers. The gels com prised a 12.5% separating gel overlaid by a 4.5% stacking gel. 20-50|Lig of sam ple p ro tein w as loaded into each well using a duck-billed Gilson tip, alongside 5ml of reconstituted prestained m olecular w eight protein m arkers (BIO-RAD kaleidoscope pre-stained markers). The gels w ere ru n at a constant voltage of 200V until the dye front had just ru n off the bottom of the gel. The gels w ere then stained in Coomassie blue stain for 1 hour at room tem perature, and de-stained overnight. For storage, gels w ere rinsed in w ater, sealed betw een two layers of cellophane (BIO-RAD Gel Air cellophane) soaked in 5% glycerol solution, and allowed to air-dry.

Acrylogel solution; 40% (w /v ) acrylamide and bis-acrylamide (37:1) Filtered and stored at 4®C.

Gel buffers:

Lower gel buffer: 1.5M Tris-HCl pH8 .8

0.4% (w /v ) SDS

Stacking gel buffer: 0.5M Tris-HCl pH6 .8 0.4% (w /v ) SDS

1 X Running buffer: 25mM Tris

0.1% SDS 192mM glycine pH8.3 Cel mixes: 8% (w /v) stacking gel: Acrylogel solution 9.375ml 20% SDS 0.15ml

Lower gel buffer 11.25 ml

H2O 9.065ml TEMED 0.0 1ml 20% (w/v) APS 0.15ml total: 30ml Acrylogel solution 2ml 20% SDS 0.05ml

U pper gel buffer 1.25 ml

H2O 6.64ml

TEMED 0.0 1ml

20% (w/v) APS 0.05ml

CHAPTER 3

Insertional M utagenesis using Restriction Enzym e-M ediated

Integration (REMI) and subsequent screening of tipless mutants

D.discoideum has a relatively small (approxim ately 50 Mbp), haploid genom e w hich consists of m ainly single copy genes and hence is highly am enable to the stu d y of d ev elo p m en tal genes by m u tag en esis. The spontaneous m utation rate per gene is approxim ately 1 0'^, i.e. a m utation will occur every 1,000,000 replications. This rate can be increased via chemical and physical m utagens. The effectiveness of these m utagens has been com pared at levels which produce equivalent survival rates (20-30%) and N 'm ethyl N '-nitro N 'nitrosoguanidine (MNNG) has been found to be the m ost m utagenic, w ith UV irrad iatio n p lu s caffeine, UV irrad iatio n alone, an d ethyl m ethane- sulphonate (EMS) causing progressively less m utagenesis [caffeine alone will only cause tem porary mutagenesis w ith m utants reverting to w ild-type after a few generations] (Liwerant and Da Silva, 1975). MNNG can be used to increase the m utation rate 1000-fold whereas UV will cause an 100-fold increase b u t will also p ro d u ce a greater p ro p o rtio n of killing relative to stable m u ta n ts (Yanagisawa et al, 1967; Sussman and Sussman, 1953). A lthough m utagenesis is easy to perform , m ultiple m utations m ay be created using these pow erful m utagens and it is impossible to determine how m any genes have been affected in any one m utant. For this reason, an aberant developm ental p h en o ty p e cannot be attributed to one particular gene.

Following the isolation of a developm ental m utant it is possible to m ap the m utated gene to a particular linkage group using parasexual genetics but this provides very limited, if any, functional inform ation about the norm al role of that gene (Newell, 1978; W elker and Williams, 1982). In addition, it is very difficult to perform m atings betw een diploid D.discoideum strains and hence observe the effects of a p articu lar m u tatio n w ith in a specific genetic background. Therefore, only a lim ited understanding of developm ent could, until recently, be gained through the stu d y of developm ental m utants since there w as no efficient m ethod by which the affected genes could be cloned and investigated directly. The developm ent of a technique w hich allow ed the sim ultaneous disruption and tagging of developm ental genes w ould therefore overcom e the lim its of conventional m utagenesis an d p e rm it a greater understanding of D.discoideum development. This is the goal to be achieved by the use of RFMI.

Restriction enzym e-m ediated integration (REMI) in D.discoideum w as developed follow ing an observation in Saccharomyces cerevisiae th at BamHI- linearised plasm id DNA will integrate into the genome at BamHI recognition sites w hen transform ed in the presence of BamHI. Specifically, the addition of BamHI p roduced a seven-fold increase in transform ation efficiency in yeast w ith approxim ately 80% of all integrations occuring at BamHI sites, as com pared to only 5% w hen transform ation was perform ed in the absence of the enzym e (Schiestl and Petes, 1991). This phenom enon is m ost sim ply explained by a sim ple ligation model in which the restriction enzym e enters the host cell at the time of transform ation along w ith the introduced DNA [for w hich there is evidence in m am m alian cells du rin g transform ation by electroporation (W inegar et al, 1989)], cleaving the genom ic DNA and th u s facilitating integration at recognition sites before the DNA is repaired. W hen attem pted in

D.discoideum , the efficiency of plasm id DNA integration was show n to increase tw enty to sixty-fold by the addition of the same restriction enzym e as used to linearise the plasm id, or one w hich produced the sam e cohesive ends; for exam ple BamHI could be replaced by SauSAI for use w ith BamHI-linearised plasm id w ith no loss in efficiency. Substitution of SauSAI, w hich has a four base-pair recognition site as opposed to the six base-pair recognition site of BamHI increased the num ber of stable transform ants recovered by one third, p resu m ab ly because there are m ore SauSAI sites w ith in the D.discoideum

genome. Sequence analysis at the points of integration show ed th at vector integrated apparently random ly w ithin the genome, w herever there w as an appropriate recognition site, and hence wide-scale application of this technique should access the entire genome. Using REMI, developm ental m utants w ere recovered at a rate of 0.04% (16 out of 6000 transform ants) and 25% of these developm ental m utants showed a tipless phenotype (Kuspa and Loomis, 1992). A lthough conventional m utagenesis can be used to create m orphogenetic m utants at a rate far in excess of this, it provides no m eans of identifying the m u ta te d genes, as ex p lain ed above. REMI, h o w ev er, facilitates this identification since the identity of the plasm id integrated into the disru p ted gene is known. The steps involved in the cloning of a developm ental gene from a m orphogenetic m utant using REMI are outlined on the next page:

1, A p lasm id carrying tw o selectable m arkers, one for selection in

D.discoideum an d one in E.Coli is electroporated into w ild-type D.discoideum

cells using REMI, causing insertional mutagenesis.

2, T ransform ants are selected and m orphogenetic m u tan ts isolated by phenotype.

3, A ran g e of restriction enzym es w hich cleave eith er o u tsid e the integration vector, or w ithin it but still m aintain the bacterial selectable marker, is u sed to digest genomic DNA p rep ared from a m u tan t of interest, and fragm ents w hich include the vector plus a few KBs of flanking genomic DNA identified by Southern blot.

4, Digested genomic DNA which includes the desired fragm ent is ligated, transform ed into E.Coli, and hence the desired fragm ent can be isolated by selection. This ligated DNA is term ed a "full-rescue" if it contains flanking DN A from either side of the insertion, or a "half-rescue" if there is DNA from only one side. If the presum ptive full-rescue is correct then Southern blot analysis of restricted wild-type and m utant genomic DNA w ith the full-rescue w ill show a restriction fragm ent length polym orphism (RFLP) in the m utant corresponding to the insertion of the plasmid, and this increased size should be the sam e as the full-rescue size.

5, To p ro v e th a t d is ru p tio n of th e id e n tifie d gene c au sed th e m orphogenetic m utation displayed, the m utant phenotype is recreated w ithin w ild -ty p e by hom ologous recom bination, ie by electroporation of the full rescue into D.discoideum w ithout restriction enzyme .

6, The flanking DNA is then sequenced using prim ers from w ithin the vector to obtain partial sequence of the disrupted gene. From this the rest of the gene sequence can be derived by screening of genomic or cDNA libraries, and hence the p ro tein sequence derived. It is then possible to investigate the structure, function, localisation and interactions of the gene and its product.

The REMI protocol used in this thesis (adapted from Kuspa and Loomis by D r L.D rury; see 2.2.2.11.1) used a Bam H I-linearised in teg ratin g vector electroporated into D.discoideum in the presence of DpnII.

This ch ap ter aim s to describe the initial screen of m orphogenetic m utants created using REMI for those defective in tip formation, and to present the d ata w hich led to the selection of tw o of these m u tan ts for fu rth er characterisation.

3.2 Tipless mutants constitute a small proportion of all REMI phenotypes.

The frequency w ith which particular classes of m utant phenotypes were createdusing REMI is show n below in table 1.

M utant phenotype decription Num ber created using REMI Frequency (%) Aggregation deficient 4 6 .6 6 Loose m ound 4 6 .6 6 Tight m ound 3 5 Multi-tip 27 45 Culmination defective 2 2 36.66 1 TOTAL 60 1 0 0

Table 3.1: Frequency of REMI phenotypes

These REMI m utants w ere isolated from 5 separate REMI events. The total n u m b er of transform ants, ie developm ental m u tan ts and ap p aren tly unaffected cells containing an integrated plasm id, w as not recorded. Since each electroporation involved 1x10? D.discoideum cells, and hence 60 developm ental m u tan ts w ere isolated from a total of 5x10? transform ed cells, the rate of form ation of m orphogenetic m utants w as 1.2x1 0"^ and of tipless m u tan ts (defined in this thesis as tight m ounds) was 6xlO'S.

Of the phen o ty p e groups u sed to classify REMI m u tan ts, tipless m u ta n ts co n stitu ted the sm allest group of developm ental m u tan ts, an d represented only 5% of all m utants created using this technique.

In addition to the three tipless m utants which I created, tw o m ore were obtained th ro u g h an exchange program of tipless for aggregation-deficient m utants w ith another laboratory which was m aking REMI m utants (Prof. Peter van H aastert, University of Groningen, Netherlands).

3.3 Southern blot analysis shows that all tipless mutants are created b y plasmid insertion at separate sites w ithin the genome

To ascertain w hether any of the tipless m utants w ere the result of the sam e m utation ie, due to the vector integrating at the same point w ith in the genom e, genomic DNA w as p rep ared from each, digested w ith a range of restriction enzymes which w ould cut both w ithin and outside of the vector and the fragm ents separated by electrophoresis through a low percentage (0.6%) TAB agarose gel. The separated fragm ents w ere transferred to nitrocellulose filters (Hybond N+) by Southern blot and the filters hybridised w ith an a-^^P- dATP labelled probe m ade from pU C8, w hich shares hom ology w ith the ampicillin resistance-containing region of the integrating vector. The resulting p attern s of restriction fragm ents w hich contained the vector are show n in Figure 3.1 overleaf.

C om parison of the restriction patterns show s that all m utants possess a separate site of insertion of the integrating vector and hence all m utants are the result of separate m utations. It is impossible to rule out the chance th at two m utants are the result of separate m utations w ithin the same gene, b u t this is extremely unlikely if the m utation frequency is considered together w ith the size of gene and arrangem ent of restriction enzym e sites required to produce tw o different Southern blots from the same gene. Since only one fragm ent containing the plasm id is detected for each separate restriction digest, this shows that there was only one plasm id integrated per m utant.

These Southern blots w ere also used to identify restriction fragm ents w hich w ould yield flanking genomic DNA from either side of the integrated vector, for half- and full-rescues.

^ i t CO CO U X UÎ ■ a - f2 X a CO CÛ U X C/5 CO W5 u a ^ ÆCO c/5 U X W5 u Û. - -Û CO w u X W5 V O. J 2 ^ CO CO CA) u X 11 12 12 10 11 TJ OJ % I—‘ o 4^ TIPA TIPB Sph I I AMP I PCM2 URA TIPC TIPD Xba I Cla I I _ I I AMP I pJBl TIPE Cla I URA Xba I h a lf rescue

Sph I h alf rescue Cla I h a lf rescue

Bgl II/ Bel 1/ Cla I fu ll recue Bgl II/ Bel 1/ Sph I fu ll recue

Figure 3.1 Southern blots of restricted tipless mutant genomic DNA used to identify suitable fragments for half and full rescue, with maps of regions of plasmid integration. [There are no Xhol sites in pCM2. This restriction enzyme was used because the original pCM2 map sent from Groningen mistakenly contained these sites.]

3.4 Some tipless mutants show an altered phenotype on different media

The five tipless m utants described in this chapter, called TIPA, B, C, D and E, were initially selected by virtue of a tipless phenotype w hen grow n in association w ith K.aerogenes on SM agar. H ow ever, some REMI m utants had previously been reported to show an altered phenotype w hen allow ed to develop on buffered (KK2) and un-buffered (H2O) non-nutrient agar. Therefore, to dem onstrate th at the developm ental arrest displayed by the five tipless m utants w as not altered u nder more stringent conditions, their developm ent on non-nutrient agar was observed and is show n in figure 3.2 overleaf.

Based upon the phenotypes displayed in figure 3.2, it seems possible to divide the m utants into two groups. The first group contains m utants TIPB, TIPC, and TIPD, all of which show an unaltered phenotype on various media. All of these show a w ide plaque growing-edge w hen grow n u pon bacteria, and form tight m ounds upon non-nutrient agar w hich do not develop beyond this stage. The other two m utants, TIPA and TIPE, form the second group. The plaques of both of these have a discrete edge w hen grow n upo n bacteria, and both show delayed aggregation, resulting in tiny loose aggregates by 30 hours of developm ent upon non-nutrient agar.

3.5 Development does not occur in the absence of morphogenesis to produce mature spores

To ascertain w hether any of these tipless m utants could produce spores despite their apparent developmental arrest, m ounds were allowed to develop u p o n unbuffered (H2O) agar for 30 hours, and then individual m ounds were lifted off the substratum and squashed under a coverslip on a glass microscope slide. The squashes w ere then exam ined m icroscopically for spore-like structures by morphology.

To test w hether any observed spore-like structures w ere true, m ature spores, defined by detergent resistance, 1x1 0^ cells of each m u ta n t w ere developed u pon w ater agar for approxim ately 30 hours, an d the m ounds treated w ith 0.3% cem ulsol. The n u m ber of m atu re spores form ed w as quantified by counting any spores th at rem ained intact after lysis, using a haemocytometer.

SM Agar H2O Agar KK2 Agar TIPA I' C: - ' TIPB é * * - I " TIPC m m m , • * « t TIPD ! r J i aP • • % t \ « i y * j ^ .. ft » \ ^ ^ » J TIPE # t f t '

Figure 3.2 Phenotypes of tipless mutantsTIPA-E on various media A ll im ages are show n at the sam e magnification. D evelopm ents on H2O (unbuffered) and KKg (buffered) agar recorded at 18 hours.

Strain Spores present w ithin terminal structure (by morphology) No. of cells surviving lysis counted Proportion of detergent resisitant cells