Yeast techniques

8.7.1 Yeast m edia and plates YPD

YPD agar

Yeast nitrogen base lacking amino acids

CSM (casamino acids lacking ura, trp, his leu)

Selective m edia

2%

Selective agar

Am inotriazole

2% Bacto-peptone, 1% Yeast extract, 2% D- glucose

As for YPD b u t w ith 2% Bactoagar (Difco). Difco 0919-15

BIO 101 4540-012

0.67% Yeast N itrogen Base w / o am ino acids D-glucose, 0.06% CSM

Then as required: 0.01% L-Leucine, 0.002% L- Histidine HCl m onohydrate, 0.002% L- Tryptophane, 0.002% L-Uracil (all w /v ) As for selective m edia b u t w ith 2% Bactoagar (Difco)

Sigma A 8056

8.7.2 Yeast strain

HF7C M A Ta, ura3-52, his3D200, Iys2,ade2-101,trpl,leu2

gal4,gal80,LYS::GALl-HIS3,

URA3::(GAL4 17-mers)^-CYCl-LacZ

8.7.3 G row th C onditions

All liquid cultures w ere grow n at 30°C at 200rpm shaking unless otherw ise indicated in either YPD or appropriate selective m edia containing 2% D-glucose. All agar cultures w ere grown at 30°C. Where indicated 3-aminotriazole w as used to increase the stringency of histidine selection.

8.7.4 Yeast transform ation

Sm all Scale T ransform ation: For plasm id transform ation 300ml culture of the ap p ro p riate strain w as grow n in YP broth + 2% D-glucose at 30°C until the OC>600nm was -0.8. The cells were pelleted centrifugation at 2000rpm for 5min , then w ashed first w ith 1 0 0 ml of distilled w ater and subsequently w ith 1 0ml of

TEL (lOm M Tris pHS.O, Im M EDTA, lOOmM L ith iu m A cetate). A fter

centrifugation at 2000rpm for 5 mins the cells resuspended in 2ml TEL. 100|il of the yeast cells w ere then mixed w ith l-3|ig of plasm id DNA in 50|xl of TE and placed at 30°C for 30min. 700 |il of TELPEG (40% w /v PEG3350 (Sigma P 3640) in TEL) w as then added to the y east/D N A m ixture and incubated for a further 60min. This w as followed by 5 m inutes heat shock at 42°C. The yeast cells were

then pelleted by centrifugation at 6500rpm for 10 seconds. The supernatant w as decanted and cells w ere resuspended in 200|xl of w ater and p lated on the appropriate selective plates and grown at 30°C incubator for 3 days.

L ibrary T ransform ation (Sequential cotransform ation): The yeast host strain w as first transform ed w ith the bait plasm id following the sm all-scale yeast transform ation protocol. One of the resulting transform ant colonies w as then used to prepare competent cells for the large-scale transform ation. O vernight 5ml culture of the yeast containing the bait plasm id was grow n in the selective media. This culture w as then used to inoculate a 300ml culture of the sam e m edia and grow n overnight at 30®C.

This 300ml culture was then used to inoculate 3000ml of selective m edia and was grow n w ith shaking until the cells reach OD^OOnm of 0.3-0.5. Cells w ere then pelleted by centrifugation at 2500rpm for 5min at room tem perature. The cells w ere resuspended pellet in 800ml of w ater and recentrifuged. The resulting pellet w as resuspended in 24ml of TEL (see above). Cells w ere incubated for lOmin at room tem perature. DNA mixture containing 1ml of lO m g/m l denatured salmon sperm DNA and 500|Xg of library plasm id DNA w as then mixed w ith the com petent yeast cells. 168ml of TELPEG (see above) w as th en ad d ed to this suspension and incubated for 30min at room tem perature. 21.2ml DMSO w as then added to this cell/D N A mix. This m ixture was heat shocked at 42°C for 6min, w ith occasional sw irling to facilitate heat transfer. Cells w ere rap id ly cooled to room tem perature in a w ater bath and pelleted by centrifugation at 2500rpm for 5m in at room tem perature. The pellet w as resuspended in 50ml of TE and recentrifuged. Supernatant w as decanted and cells resu sp en d ed in 1000ml of YPD m edium and incubated for Ih r at 30®C, w ith shaking at 180rpm. Cells were pelleted by centrifugation at 2500rpm for 5min and w ashed again w ith 50ml of TE. The final pellet of cells w as resuspended in 20ml of TE. 1ml aliquots w ere then spread on 20cm square plates on agar lacking histidine, tryptophan, and leucine. Plates were left to grow for 4 days before the colonies were picked for fu th er analysis. To obtain an indication of the p rim ary transform ation efficiency, lOjxl of the transform ation m ixture w as p lated on agar lacking tryptophan and leucine.

8.7.5 Isolation of plasm id DNA from yeast

Plasm id DNA was rescued from 2ml of exponentially grow ing yeast. The yeast w ere pelleted and resuspended in 200pl of lysis solution (lOOmM NaCl, lOmM Tris pH 8, Im M EDTA, 2% TritonXlOO, 1% SDS) before a d d in g 200|Lil of pheno h ch lo ro fo rm (1:1 v /v ) and 0.3g of acid w ash ed glass bead s (425- bOOmicrons - Sigma G 8772) and vortexing for 2 m inutes. After centrifugation for 2 m inutes at 13000 rpm the upper phase was transferred to a fresh tube and 2.5

volum es of ethanol and 0.1 volum e of 3M NaAc pH5.2 w as added. After centrifugation for 10 m inutes at 13000 rpm the pellet w as w ashed w ith 70% ethanol and resuspended in 50 jil of TE. The DNA was electroporated into D H 5a bacteria (see Section 8.3).

8.7.6 p-Galactosidase assay in yeast

L iquid assay: Yeast cells were grow n until they reached OD600nm of 0.5-1.0) (5 individual transform ants were grow n for each assay ). The cells w ere pelleted by centrifugation at 2000rpm for 5min, w ashed w ith water and resuspended in 200|xl of O.IM TrisCl pH7.5, 0.5% Triton XlOO. After rapid freezing in dry ice, the cells w ere thaw ed at room tem perature and pipetted up and down. lOOpl of this cell suspension w as then added to a 1.5ml m icro-centrifuge tube containing 0.5ml LacZ buffer (60mM Na2HP04.7H20, 40mM NaH2P04, lOmM KCl, Im M M gS04, 2 .7 m l/l 6-m ercaptoethanol) and 0.1ml O N P G (4m g/m l). Cells w ere then incubated at 37°C until the reaction mix tu rn ed yellow, reactions w ere then quenched by addition of 250jj,l of IM N a2CCb; and the reaction time w as noted.

Tubes w ere then centrifuged at 13000rpm for 2 m inutes. The OD420nm of the supernatant w as m easured and the pellet were resuspended in 1ml of w ater and OD600nm w as then m easured, fi-galactosidase activity was m easured using the following equation. OD420nm/(OD^OOnmx reaction time).

F ilter assay: Yeast colonies w ere streaked on 82mm H ybond-N nylon filters placed on top of the selective agar plates and grown overnight. The m em brane w as then rem oved form the plate and subm erged in liquid nitrogen for 10-15 seconds. Filter w as then air dried and put, colony side facing up, on a 3MM W hatm an p ap er soaked in 1.8ml lacZ buffer (see above) and 25|il of X-Gal (Calbiochem 203782 - 2 0 m g /m l in Dim ethy 1-formamide). The filter was then incubated at 30°C until the colonies turned blue which took betw een 20 m inutes - 3hrs. The reactions were quenched by p u ttin g the filter on a 3MM W hatm an soaked in lMNa2C03.

8.7.7 Effector m utant screen

An HF7C strain carrying pGADPKN was transform ed w ith pGBT9RhoAlit)rary*

and 400 in d ependent transform ants were screened for HIS3 expression. The pGBTRhoA plasm ids w ere rescued from 31 PKN interaction positive colonies by grow ing 2ml exponential liquid cultures, these w ere pooled and the plasm id rescued by pelleting cells, resuspending in 3ml lysis solution (lOOmM NaCl, lOmM Tris pH8, ImMEDTA, 2% TritonXlOO, 1% SDS), 3ml phenohchloroform (1:1 v /v ) , and 4g acid w ashed beads (425-600microns - Sigma G 8772) and vortexing for 2mins. After centrifugation the aqueous phase w as rem oved and DNA precipitated in 1/10 volum e 3M NaAc pH5.2 and 2.5 volum es ethanol.

Before transform ing into D H 5a the plasm id mix w as digested w ith Clal to linearise the pGADPKN plasm id. Bacterial transform ants w ere pooled and retransform ed into yeast carrying pGADROCK; 107 transform ants w ere screened for lack of grow th on plates lacking histidine b u t containing Im M aminotriazole.

pGBT9RhoAl^t>rary* plasm ids were isolated as before from 36 ROCK interaction

negative colonies. After transform ation into D H 5a, plasm ids w ere prepared from individual D H 5a colonies and checked by retransform ation into yeast and

sequencing. In a second screen 402 colonies carrying pGADROCK and

pGBT9RhoAlit>rary* were screened for lacZ expression (strong blue colour w ithin

three hours; assay described Clontech catalogue), the RhoA m u tan t plasm ids

w ere rescu ed from 68 ROCK in teractio n p ositive clones. These w ere

subsequently transform ed into yeast carrying pGADPKN; 84 colonies w ere screened and pGBT9RhoA m utant plasm id was rescued from 16 colonies unable to grow on plates lacking histidine. These m utants were checked by sequencing and retransform ation (as above).