Two Hybrid Protocols

pAS1 HA Epitope and Polylinker Reading Frame

pAS1 HA epitope and polylinker reading frame

R1 NheI

GAA TTC ATG GCT TAC CCA TAC GAT GTT CCA GAT TAC GCT AGC TTG GGT GGT

SfiI .

CAT ATG GCC ATG GAG GCC CCG GGG ATC C

NdeI NcoI SmaI BamHI

pACT2 HA epitope and polylinker

NheI

ATG GCT TAC CCA TAC GAT GTT CCA GAT TAC GCT AGC TTG GGT GGT

SfiI .

CAT ATG GCC ATG GAG GCC CCG GGG ATC CGA ATT CGA GCT CGA GAG ATC TAT

NdeI NcoI SmaI BamHI R1 SacI XhoI BglII

GAATCGTAGATACTGAAAAACCCCGCAAGTTCACTTCAACTGTGCATCGTGCACCATCTC

AATTTCTTTCATTTATACATCGTTTTGCCT

Sequence around the cloning site of pACT (pSE1107)

Mlu I

GCGTATAACGCGTTTGGAATCACTACAGGGATGTTTAATACCACTACAATGGATGATGT

ATAT AAC TAT CTA TTC GAT GAT GAA GAT ACC CCA CCA AAC CCA AAA AAA

Bgl II BamH Bgl II

GAG ATC TGG AAT TCG GAT CCT CGA GAG ATC TAT

EcoRI XhoI

GAATCGTAGATACTGAAAAACCCCGCAAGTTCACTTCAACTGTGCATCGTGCACCATCTC

AATTTCTTTCATTTATACATCGTTTTGCCT

Sequencing primers

pACT forward 5′ 5′ CTATCTATTCGATGATGAAG

pACT Reverse 3′ 5′ ACAGTTGAAGTGAACTTGCG

GAL Promoter Forward 5′ TACTTTAACGTCAAGGAGAA

lacZ 5′ agctggcgtaatagcgaagag

(Reverse primer for Lambda ADH and YES)

ADH Forward 5′ TCTGCACAATATTTCAAGCT

The sequences of the PCR primers we have used successfully are:

1)

TAA TAC GAC TCA CTA TAG GGA GAC CAC ATG GAT GAT GTA TAT AAC TAT CTA TTC

T7 Promoter Met Gal4 Activation Domain

21 AA before the BglII site

2)

CTA CCA GAA TTC GGC ATG CCG GTA GAG GTG TGG TCA

In the ADH Terminator

Protein Interaction Cloning Using the Two Hybrid System

Library transformation using Y190

Y190 = MATa gal4 gal80 his3 trp1-901 ade2-101 ura3-52 leu2-3,-112

+ URA3::GAL–> lacZ, LYS2::GAL–>HIS3 cyhr

After subcloning the gene of interest into pAS1-CYH or a similar GAL4 DNA-binding domain fusion vector, transform this construct into Y190 selecting for TRP1 in the case of pAS1. It should be noted that expression of the fusion protein should be verified by western blotting with anti HA antibodies available from BABCO if specific antisera are not available for your protein. Occasionally, a bona fide fusion is made and cannot be detected with HA (levels too low, but sufficient for screening).

The resulting strain should be checked for its growth properties on SC-His plates containing differing concentrations of 3-AT (3-aminotriazole, SIGMA, A8056) and on its ability to activate the lacZ reporter. These tests should be carried out relative to strains carrying pSE1112 alone. We have found that 3-AT concentrations of 25 mM to 50 mM are sufficient to select against pAS1 subclones that fail to activate transcription on their own. If your construct activates transcription alone, it cannot be used in this assay. If it fails to activate transcription, like most fusions, you may proceed to the library transformation step. Also check out the RNA transformation protocol we have included. It works a little more reproducibly in our hands.

1. Use a colony or overnight to inoculate 200 mls SC-Trp and grow overnight at 30oC. The use of a saturated overnight from the night before ensures that the 200 ml culture will be fully grown the next day.

2. Take OD A600 of above culture and inoculate 500 mls YEPD such that in ~2 generations

(3- 4 hrs) the A600=0.5 to 0.8 . (Synthetic complete – Trp media is used to select for pAS1 but YEPD gives best transformation efficiencies)

3. Harvest cells at 5K for 10 minutes in a Sorvall centrifuge

4. Wash once with distilled water ~100 mls and resuspend in 50 mls LiSORB and incubate at 30oC for 15-30′.

5. Spin down as above and resuspend in ~625 µl LiSORB. Hold on ice.

6. Prepare carrier DNA mix.

Boil 200 µl 20 mg/ml sheared salmon sperm DNA for 7-10′

add 800 µl LISORB (room temp, RT)

mix by pipeting mixture up and down

cool to RT (ice can be used,but care must be taken to ensure that the Temp. does not go

lower than RT or the mixture will gel

add 40µg library DNA

7. Add ~100µl of above DNA mix to 100 µl cells from step 6.

8. Incubate at 30oC for 30 minutes (optional)

9. To 100 µl cells + DNA add 900 µl 40% PEG3350 in 100 mM LiAc/TE and incubate at 30oC for 30′. Heat Shock at 42°C for 7 minutes. Plate 5 ul of cells to test transformation efficiency on SC-Trp, Leu.

10. Recovery: Pool cells and add to ~100ml SC–His,Trp, Leu liquid media, shake at 30oC for 1-3 hours; harvest cells and resuspend in ~6 mls of SC–His,Trp,Leu liquid media and plate ~300 µl per 150mm plate (SC–His,Trp, Leu+ 25 mm AT) or 50 mM 3-AT. (Plating directly from the PEG also works but is more messy.)

11. Colonies that grow after 3 to 5 days are then tested for b-galactosidase activity using the X-Gal colony filter assay described in the accompaning protocol. Blue colonies are taken for further study, they can often be taken directly from the filters in addition to the original plate.

Efficiency = ~5×104 to 105 colonies/µg cDNA library.

For determination of efficiency plate 5 µl before and after recovery on SC-Trp, Leu

LiSORB=100 mM LiOAC, 10 mM Tris pH 8, 1 mM EDTA, 1 M Sorbitol.

LiAcTE=100 mM LiOAC, 10 mM Tris pH 8, 1 mM EDTA

We have found that total yeast RNA works more reproducibly as a carrier but it is more work to prepare the RNA than DNA. That transformation protocol is included as well. We usually place the transformation mix in SC-His, Trp, Leu after the heat shock step and allow it to recover for 3 hours. This allows the transformants to be established and HIS3 transcription to be activated. The cells at this stage can be pelleted and resuspended in a smaller volume and plated directly or made 10% DMSO, and frozen at -70°C. Cells in PEG seem more fragile and often die when pelleted so the recovery step is useful. Cells lose less than half their viability when frozen and can be stored indefinitely. This is useful because they can be thawed and plated at a optimal density for screening/selection at your leisure. This protocol can be scaled up for 1 liter of cells or more.

The HIS3/3-AT selection sometimes works as a good selection and sometimes looks like more of an enrichment. We often see many micro colonies on the original selection plates. Occasionally they are 1% of the total Leu+Trp+ colonies. In most cases true positives continue to grow into large colonies while the micro-colonies seem to stop growing. The secondary screen for blues eliminates these micro colonies. The majority of His+ blue colonies are the large colonies that grow out. An enrichment of 100-fold is very useful because it allows you to screen 100 times as many colonies on a single plate so a whole library can be screened in only 20 large plates. We have also developed a GAL–>URA3 selection system which requires higher levels of 2 hybrid activated transcription than the His selection. That strain is available upon request.

Included in the kit is pSE1111 (SNF4 fused to the activation domain in pSE1107) and pSE1112 (SNF1 fused to the DNA-binding domain of GAL4 in pAS1). These are both inserted as BamH1 fragments. These can serve as a positive control for X-gal staining and 3-AT resistance.

It should be noted that pAS1 alone can activate lacZ weakly. Therefore it is not a good negative control. pSE1112 is a better negative control. The weak activation of pAS1 appears to go away when genes are cloned into it. We do not understand why it is weakly activating alone, but we think it is likely due to sequences beyond the polylinker and which are of no consequence once cDNAs are cloned into it.

We have recently made improvements in the system. We have constructed a Y153 derivative, Y190, that is resistant to cycloheximide (2.5 ug/ml) due to a mutation in the CYH2 gene. Y190 is now being supplied with the kit. This is a recessive drug resistance. When a plasmid carrying the wild type CYH2 gene is in the strain, cells become sensitive to cycloheximide. We have constructed a pAS1-CYH plasmid that contains the CYH2 gene. After a positive clone has been selected in the system, loss of the pAS1-CYH plasmid can be achieved by streaking on SC-Leu 2.5 ug/ml cycloheximide media. It is probably a good idea to streak the colonies out on SC-Leu before streaking on cycloheximide media to allow plasmid loss and dilution of the CYH2 gene product. However, it does work streaking directly from SC-TrpLeu. The colonies that grow should be Trp-, but they should be checked for loss of the TRP marker, just to be safe and avoid CYH2 gene conversion events. This plasmid loss allows one to check for plasmid dependency of lacZ activation as well as generating a strain that contains only the library plasmid, facilitating plasmid recovery into bacteria.

False positives do occur that appear to be dependent upon both plasmids. This is a reoccurring problem of the 2-hybrid system. You should give some thought to secondary criteria for distinguishing a true positive. False positives will light up many non-related pAS1 fusions, that is one of the definitions of a false positive. To eliminate false positives, we usually generate a strain that has lost the pAS plasmid but retains the library plasmid. This is done using the CYH trick or growing in YEPD, plating on SC-Leu and replica plating to SC-Trp to look for loss of pAS1. This strain (Leu+Trp-) is mated to a strain,Y187 = MATa gal4 gal80 his3 trp1-901 ade2-101 ura3-52 leu2-3,-112 URA3::GAL–>lacZ, that is of the opposite mating type but contains different unrelated fusions in pAS1(Leu-Trp+) such as SNF1(pSE1112), lamin, p53 (Lamin and p53 are provided by Stan Fields). Once your Y190 strain of interest has lost the pAS1-fusion plasmid (by plasmid loss or cycloheximide selection) it is Trp-Leu+ and can be mated to Y187 containing pAS1-X. Diploids can be selected by growth on SC-TrpLeu and then immediately tested for b-galactosidase activity in the filter screen assay. Colonies that activate lacZ expression significantly above background levels (pSE1112) probably contain Leu plasmids encoding false positives that non-specifically activate your fusion and should be disgarded.

We have used several different tests to detect in vitro binding of positives. One is to make a PCR primer to the library plasmid that has a T7 promoter placed in an appropriate position to place the insert of the library plasmid under T7 control. The PCR-derived fragments can then be directly added to a coupled transcription-translation system (TnT from Promega) and radiolabeled protein made. We usually add 6 ul of a robust 30 cycle PCR reaction to 25 ul of the translation mix(TnT). 5 ul of this reaction mixture run on a SDS gel gives a readily detectible signal on an overnight exposure. This tells you the size of the fused protein and can be used to detect interaction in vitro with a GST-bait fusion.

The sequences of the PCR primers we have used successfully are:

1)

TAA TAC GAC TCA CTA TAG GGA GAC CAC ATG GAT GAT GTA TAT AAC TAT CTA TTC

T7 Promoter Met Gal4 Activation Domain

21 AA before the BglII site

2)

CTA CCA GAA TTC GGC ATG CCG GTA GAG GTG TGG TCA

In the ADH TerminatorA second test is to IP your protein out of yeast extracts and then use antibodies to the activation domain of GAL4 to detect binding of the fusion protein. Our GAL4 activation domain antibodies are currently being tested for this purpose. Unfortunately, they do not IP presently.

A third method is to switch the bait and prey in their respective plasmids, i.e. take the library insert out of pACT and insert it into pAS1, and place the original pAS1 insert into pACT. The majority of false positives will not interact in this test. It should be noted that some true positives may not activate for structural reasons, so only a positive result can be trusted. We have recently placed the pAS1 polylinker into pACT to facilitate this transfer, creating pACT2.

For general yeast protocols we recommend the Methods in Enzymology Vol. 194 “Guide to Yeast Genetics and Molecular Biology” by Guthrie and Fink and the Red book.

As with all protocols, this is probably not optimal and I encourage you to add variations. Let me know if any new variation makes for a simpler or more efficient protocol. For example, several people have said that direct plating of the transformation mix in peg onto selective plates workes well for them. It is probably worth a try.

Our lab, Wade Harper’s lab, and Stan Fields’ lab have all successfully isolated clones from our l ACT libraries using the HIS3 selection of Y153 and Y190 and have each contributed to these protocols. Good luck.

Lambda ACT + YES

Enclosed you will find a cDNA library made in the lambda vector lYES-R or ACT (activation domain) and several other strains depending upon your request. The titer should be 1 x 109 PFU/ml or greater, with between 1 x 107 and 1 x 108 total recombinants, amplified only once from the packaging. Upon reciept, these phage should be titered, amplified to give a higher titer stock if necessary, and aliquotes can be frozen (9% DMSO, -70°C) for future use including DNA preparation. Libraries should be titered and amplified as phage on LE392 or equivalent bacterial host (NOT BNN132).

The cDNA was sized selected to be >600bp. The bacterial strain is BNN132 = JM107/lKC, a kanr lambda lysogen containing the cre gene. Infection of this strain with the library will produce Ampr colonies that have quantitatively excised the plasmid via cre-mediated lox recombination. I typically infect the library into log phase JM107/lKC cells and plate about 108 infected cells (108 phage in 3 x 108 cells) per large LB Amp-50 ug/ml plate (add 0.2% glucose if using lYES-R). I usually absorb the phage for 30′ at 30°C and then add LB and allow the cells plus phage to grow for an hour before plating. This allows the cells to express the bla gene and probably to undergo recombination. Since lYES-R was designed to express both in E. coli and yeast, I use the glucose to help repress the lac promoter (JM107 is also lacIq). This helps prevent biasing of the library. lACT does not contain the lac promoter so glucose is not required. I usually plate about 10 large plates worth of library which is about 100 times the original number of recombinants. I have found no difference between plating at 30°C or 37°C. The lKC phage has a wild type repressor. DNA can be prepared directly from the lysogens scraped from these plates. I usually take these cells and resuspend them in terrific broth and grow them up a little further to get more DNA (6 liters gives 2 mg of plasmid DNA). Remember, these are pBR322 based plasmids so the copy number is not high. There is no real reason not to do the whole thing in liquid. I think that growth on plates presents less of a competitive situation for the clones, but it may not really be needed in actuality. For further information I suggest you read PNAS 88:1731-1734 on the lYES-R system. Two other publications concerning the 2-hybrid system are Genes and Dev. 7:555-569. and JBC 268:4608-4611.

Although this goes without saying, these libraries and strains should not be distributed to other researchers without my prior consent and should be used for only the experiments you described in your letter. Good luck.

SOS Recruitment System (SRS) Strains

cdc25-2 MATa (ura3 lys2 leu2 trp1 cdc25-2 his3-D200 ade101 GAL+)

Temperature sensitive yeast strain; grow on YPD plates at 24°C.

Lambda cloning vector.

Lambda MS-Trp Bacteria harboring lMS-TRP as an extra chromosomal plasmid lysogen. This vector is pMS-TRP inserted via the NotI site into in the arms of lYES and can be excised by cre-mediated site-specific recombination in BNN132. This vector DNA can be prepared by CsCl gradient methods for use as a cloning vector for the production of cDNA libraries. cDNA can be inserted non-directionally into the XhoI or EcoR1 sites or directionally using the EcoR1-XhoI sites and cDNA prepared using the kit from Stratagene. Grow this lysogen in LB Amp (50 µg/ ml) at 24°C because it has the temperature sensitive cI857 mutation.

The following plasmids are in bacteria and should be grown on LB Amp (100 µg/ ml) plates at 37°C:

pMS-TRP (TRP1) This expression vector has the myristoylation signal under GAL control and is used for library production

pSOC4 (URA3) SOS expression vector with polylinker at C terminus for fusions

pSON8 (URA3) SOS expression vector with polylinker at N terminus for fusions

pMS-TRP-FOS (TRP1) Positive control; FOS with myristoylation signal

pSOC4 JUN (URA3) Positive control; JUN fused to C- terminus of SOS

pSON8 JUN (URA3) Positive control; JUN fused to N- terminus of SOS

Please streak these strains onto the appropriate plates as soon as you receive them. You can then inoculate them from single colonies into the appropriate liquid media. Freeze aliquots of overnight cultures containing 9% DMSO for future use.

Once temperature-resistant colonies are identified in a library transformation, 5-FOA can be used to select for the loss of the bait plasmid to test for plasmid dependency of the growth phenotype. Loss of the URA3 plasmid will also facilitate recovery of the library plasmid from yeast.

X-Gal Colony Filter Assay From Pierre Chevray

1. Label Scheicher and Schuell BA85 45um circular nitrocellulose filters (cat. # 20440) with a ball point pen.

2. Lay the filter onto the plate of yeast colonies and allow it to wet completely and place orientation markers on filters and plates with India ink and a needle .

3. Lift the filter off of the plate carefully to avoid smearing the colonies and place the filter in liquid nitrogen to permeabilize the cells. Five to ten seconds is sufficient. Filters can sither be submerged in the liquid nitrogen or placed on an aluminum foil float.

4. Carefully remove the filters from the liquid nitrogen ( frozen filters become very brittle) and place cell side up in a petri dish that contains 3MM chromatography paper soaked with 0.30ml/square inch of Z Buffer containing 1mg/ml X-Gal.

5. Incubate at 30oC for minutes to overnight for development of color. Cover the plate to prevent evaporation.

6. Putative positives can be picked directly off of the nitrocellulose filter if streaked immediately. All putative positives should be tested a second time to confirm.

Z Buffer for one liter

Na2HPO4 7H2O 16.1g

NaH2PO4 H20 5.5g

KCl 0.75g

MgSO4 7H2O 0.25g

2-mercaptoethanol 2.7ml

Adjust pH to 7.0.

Stock solution of X-Gal is dissolved in DMF at 100mg/ml

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