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MATCHMAKER GAL4 Two-HybridVectors Handbook

(PT3062-1)

Information Supplement for:

Catalog # Product

K1605-1 MATCHMAKER Two-Hybrid System

K1604-1 MATCHMAKER Two-Hybrid System 2

(many) MATCHMAKER Libraries

FOR RESEARCH USE ONLY

(PR6X890)

MATCHMAKER GAL4 Two-Hybrid Vectors Handbook CLONTECH Laboratories, Inc.

page Technical Service TEL: 415-424-8222 or 800-662-CLON Protocol # PT3062-12 FAX: 415-424-1064 or 800-424-1350 Version # PR6X890

I. Introduction 3

II. GAL4 Activation Domain (AD) Cloning Vectors 5

pACT 5

λACT 6

pACT2 7

pGAD10 8

pGAD424 9

pGAD GH 10

pGAD GL 11

III. GAL4 DNA-Binding Domain (DNA-BD) Cloning Vectors 12

pAS2 12

pAS2-1 13

pGBT9 14

IV. Control Plasmids 15

pCL1 15

pVA3 16

pVA3-1 16

pTD1 17

pTD1-1 17

pLAM5' 18

pLAM5'-1 18

V. References 19

VI. Plasmid and System Ordering Information 20

APPENDIX. Additional MATCHMAKER Plasmid Information 21

List of Tables

Table I. Yeast Promoter Constructs in the MATCHMAKER GAL4 Two-Hybrid Cloning Vectors 4

Table II. List of Abbreviations 4

Table III. MATCHMAKER GAL4 Two-Hybrid System Cloning Vectors 21

Table IV. MATCHMAKER GAL4 Two-Hybrid System Reporter Plamids 22

Table of Contents

Protocol # PT3062-1 Technical Service TEL: 415-424-8222 or 800-662-CLON pageVersion # PR6X890 FAX: 415-424-1064 or 800-424-1350 3


I. Introduction

This Handbook provides vector maps, MCSs, transformation markers, references, GenBank accessionnumbers (where available), and other pertinent information for the cloning and control plasmids used inthe yeast GAL4-based MATCHMAKER Two-Hybrid Systems and Libraries. For ease of use andcomparison of the plasmids, much of this information is summarized in the Tables in the Appendix.Complete sequence information for most of the plasmids is available by request from CLONTECH'sTechnical Support Dept. Selected vector sequences are accessible through our worldwide web site(http://www.clontech.com).

All of the cloning and control plasmids are shuttle vectors that replicate autonomously in both E. coli(using the Col E1 or pBR322 origins of replication) and in S. cerevisiae (using the 2µ ori). AllMATCHMAKER plasmids carry the β-lactamase (bla) gene, which confers ampicillin resistance (Ampr)in E. coli, and a nutritional gene (TRP1 or LEU2 ) that allows yeast auxotrophs transformed with theplasmid to grow on synthetic defined medium lacking tryptophan or leucine, respectively. The nutritionalmarkers also allow selection in certain auxotrophic strains of E. coli on M9 minimal medium.

The cloning plasmids generate fusion proteinsAll of the cloning plasmids provided in the MATCHMAKER Two-Hybrid Systems are used for theconstruction and expression of hybrid (fusion) proteins. The GAL4 AD plasmids generate a hybrid thatcontains the sequences for the yeast GAL4 activation domain (a.a. 768–881) and a cloned protein orcDNA library insert. The GAL4 DNA-BD plasmids generate a hybrid that contains the sequences for theGAL4 DNA-binding domain (a.a. 1–147) and a cloned protein, most often used as the bait protein in atwo-hybrid library screening. These vectors have unique restriction sites located in the MCS region atthe 3' end of the open reading frame for either the DNA-BD or the AD sequence.

For the construction of a specific hybrid, the gene encoding the protein of interest is inserted into the MCSin the correct orientation and reading frame such that a hybrid protein is generated. In the case ofMATCHMAKER Libraries, the inserts may be cloned into the MCS either directionally or nondirectionally(check the Product Analysis Certificate and the User Manual for detailed information on how the librarywas constructed). The fusion proteins are expressed in yeast cells from the full-length ADH1 promoteror a modified form of it. See Table I for the relative expression levels observed for the different plasmids;for further information on the promoters, see the Yeast Protocols Handbook (YPH), Chapter II. In allplasmids, transcription is terminated at the ADH1 transcription termination signal.

The hybrid proteins are targeted to the yeast nucleus by nuclear localization sequences. In the GAL4DNA-BD cloning plasmids, the nuclear localization sequence is an intrinsic part of the DNA-BD (Silveret al., 1984) and is not specifically indicated on the vector maps. In the GAL4 AD cloning plasmids, thenuclear localization sequence from SV40 T-antigen has been cloned into the vector between the ADH1promoter and the AD sequence. (For detailed information regarding the construction of this sequence,see Chien et al., 1991.)

Some of the cloning vectors also encode the influenza hemagglutinin (HA) epitope in the junctionbetween the GAL4 sequence and the MCS; if present, this element is indicated on the vector map. TheHA epitope tag is useful for detecting expressed fusion proteins on Western blots using an anti-HAantibody. GAL4 AD and DNA-BD Monoclonal Antibodies (#5398-1 and 5399-1, respectively) areavailable from CLONTECH for probing Western blots when using medium- to high-expression levelcloning vectors (see Table I). The DNA-BD plasmids pAS2 and pAS2-1 (Harper et al., 1993) carry thewild-type yeast CYHS2 gene, which confers sensitivity to cycloheximide in transformed cells. CYHS2 canbe used to isolate AD/library plasmids in yeast segregants after a two-hybrid library screening, and thusfacilitates the elimination of false positives.



I. Introduction continued

TABLE I. YEAST PROMOTER CONSTRUCTS IN THE MATCHMAKER GAL4 CLONING VECTORS

Regulation/ SignalRelative Protein Strength on

Vectors Promoter Expression Level Western blot a

pGAD GH, pAS2, pAS2-1 ADH1 (full-length) Ethanol-repressedb/High +++

pACT2, pACT ADH1 (truncated+)c Constitutive/medium ++

pGAD GL ADH1 (truncated) Constitutive/low +/– (weak)

pGAD424, pGAD10, pGBT9 Constitutive/ very low (not detectable)

a Soluble protein extracts were prepared from CG-1945 transformed with the indicated plasmid. Samples equivalent to ~1 OD600unit of cells were electrophoresed and then blotted to nitrocellulose filters. The blots were probed with either GAL4 DNA-BDmAb (0.5 µg/ml; #5398-1) or GAL4 AD mAb (0.4 µg/ml; #5399-1), followed by HRP-conjugated polyclonal goat anti-mouse IgG(Jackson Immunological Research; diluted 1:15,000 in TBST). Signals were detected using a chemiluminescent detectionassay and a 2.5-min exposure of x-ray film. Signal intensities were compared to that of known amounts of purified GAL4 DNA-BD or GAL4 AD.

b Transcription is repressed in late lag phase by the ethanol that accumulates in the medium as a by-product of yeast metabolism.c The truncated ADH1 promoter in pACT2 is adjacent to a section of pBR322 which acts as a transcriptional enhancer in yeast.

TABLE II. LIST OF ABBREVIATIONS

AD GAL4 activation domain (a.a. 768–881)

DNA-BD (or BD) GAL4 DNA-binding domain (a.a. 1–147)

P promoter

T transcription termination sequence

HA hemagglutinin sequence

AD/library plasmid Plasmid encoding a fusion of theGAL4 AD and a library insert



II. GAL4 Activation Domain (AD) Cloning Vectors

Figure 1. pACT map and MCS. Unique sites are in bold. pACT (Durfee et al., 1993) is used to generatea hybrid containing the GAL4 AD (a.a. 768–881) and another protein of interest (or a protein encodedby a cDNA in a fusion library). The hybrid protein is expressed at medium levels in yeast host cells fromthe enhanced, truncated ADH1 promoter and is targeted to the yeast nucleus by the SV40 T-antigennuclear localization sequence (▲ ; Chien et al., 1991). pACT contains the LEU2 gene for selection in Leu–

auxotrophic yeast strains. The Bgl II (*) sites can be used as a unique cloning site. pACT Libraries fromCLONTECH are constructed in λACT; recombinant plasmids are released by Cre-lox recombination(see Figure 2). No further pACT sequence information is available.

AAT ACC ACT ACA ATG GAT GAT GTA TAT AAC TAT CTA TTC GAT GAT GAA GAT ACC CCA

CCA AAC CCA AAA AAA GAG ATC TGG AAT TCG GAT CCT CGA GAG ATC TAT GA

A TCG TAG ATA CTG AAAAACCCCGCAAGTTCACTTCAACTGTGCATCGTGCACCATCTC

Xho IBamH IEcoR I Bgl II*Bgl II*

GAL4 AD Sequencing Primer

STOP (ORF 1)

STOP (ORF 3)

STOP (ORF 2)

MATCHMAKER 3' AD LD-Insert- Screening Amplimer

MATCHMAKER 5' AD LD-Insert Screening Amplimer

GAL4 AD

MCS Bgl II*

EcoR I BamH I Xho I Bgl II*

EcoR I (1)

Xba I (0.7)

Hpa I (1.7)

Cla I (2.7)

EcoR I (3.2)

EcoR V (3.3)

Aat II (7.5)

pACT 7.65 kb

Col E1 ori

Ampr

PADH1

2 µ ori

TADH1

LEU2∆

GAL4 AD

♦

Not I Not I

loxP



II. GAL4 Activation Domain (AD) Cloning Vectors continued

Figure 2. Conversion of a recombinant λACT to the corresponding pACT. The λACT MCS is locatedwithin an embedded plasmid, which is flanked by loxP sites at the λ junctions. Transduction of a λACTlysate into E. coli strain BNN132 promotes Cre recombinase-mediated release and recircularization ofpACT at the loxP sites. Note that the resulting plasmid contains only one loxP site.

λ Left Arm (27.4 kb) λ Right Arm (15.2 kb)

pACT

λACT (42.6 kb)

Clone cDNA into MCS of λACT

High-efficiency, single-step conversion via site-specific recombination at loxP sites (in E. coli BNN132)

loxP loxP

MCS

MCS with insert

loxP loxP

pACT 7.65 kb

Col E1 ori

Ampr

PADH1

2 µ ori

GAL4 AD

TADH1

LEU2

∆

insert

♦loxP




Figure 3. pACT2 map and MCS. Unique sites are in bold. pACT2 (Li et al., 1994) is used to generatea hybrid containing the GAL4 AD (a.a. 768–881), an HA epitope tag (Durfee et al., 1993), and anotherprotein of interest (or a protein encoded by a cDNA in a fusion library). The hybrid protein is expressedat medium levels in yeast host cells from an enhanced, truncated ADH1 promoter and is targeted to theyeast nucleus by the SV40 T-antigen nuclear localization sequence (▲ ; Chien et al., 1991). pACT2contains the LEU2 gene for selection in Leu– auxotrophic yeast strains. The Bgl II (*) sites can be usedas a unique cloning site, but this will remove the HA epitope. pACT2 libraries from CLONTECH areconstructed in λACT2; recombinant plasmids are released by Cre-lox recombination (see Figure 2).GenBank Accession: #U29899.

AAT ACC ACT ACA ATG GAT GAT GTA TAT AAC TAT CTA TTC GAT GAT GAA GAT ACC CCA CCA

AAC CCA AAA AAA GAG ATC TGT ATG GCT TAC CCA TAC GAT GTT CCA GAT TAC GCT AGC TTG

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

A TCG TAGATACTGAAAAACCCCGCAAGTTCACTTCAACTGTGCATCGTGCACCATCTC

Bgl II*

STOP (ORF 1)

STOP (ORF 3)

STOP (ORF 2)

5095 •

5035

•

4976

•

5155 •

Sma I/ Xma I

Nco I BamH I EcoR INde I

Sfi I

Xho I

Tyr Pro Tyr Asp Val Pro Asp Tyr A la

HA epitope

Sac I

Bgl II*

GAL4 AD

GAL4 AD MATCHMAKER 5' AD LD-Insert

Screening AmplimerGAL4 AD Sequencing Primer

MATCHMAKER 3' AD LD- Insert-Screening Amplimer

pACT2 8.1 kb

MCS

(5082–4981) * Bgl II

HA epitope Nde I Sfi I

Nco I Sma I Xma I

BamH I EcoR I

Sac I

Xho I * Bgl II

Nde I (235)

Nde I (6131)

Not I (4163)

Cla I (2626)

Hpa I (1581)

Xba I (598)

2 µ ori

GAL4 AD

PADH1

TADH1

LEU2

Col E1 ori

Ampr

Not I (4345)

loxP

HA

∆

Hind III (4739)

Hind III (5497)

♦




Figure 4. pGAD10 map and MCS. Unique sites are in bold. pGAD10 (Bartel et al., 1993) is used togenerate a hybrid containing the GAL4 AD (a.a. 768–881) and another protein of interest (or a proteinencoded by a cDNA in a fusion library). The hybrid protein is expressed at low levels in yeast host cellsfrom a truncated ADH1 promoter and is targeted to the yeast nucleus by the SV40 T-antigen nuclearlocalization sequence (▲ ; Chien et al., 1991). pGAD10 contains the LEU2 gene for selection in Leu–

auxotrophic yeast strains.GenBank Accession: #U13188.


CCA AAC CCA AAA AAA GAG ATC TCT CGA GGA TCC GAA TTC CAG ATC TAT GA

A TCG TAGATACTGAAAAACCCCGCAAGTTCACTTCAACTGTGCATCGTGCACCATCTC

Xho I BamH I EcoR I Bgl II*Bgl II*

GAL4 AD

STOP (ORF 1)

STOP (ORF 3)

STOP (ORF 2)


MATCHMAKER 5' AD LD-Insert Screening Amplimer755

•

812 •

862 •


2 µ ori

GAL4 AD

pGAD10 6.65 kb

PADH1

TADH1

LEU2

Col E1 ori

Ampr

Sph I (10)

Hind III (410)

Kpn I (484)

Mlu I (727)

Hind III (1096)

Sph I (1424)

EcoR V (1882)

Cla I (2478)

Bgl I (4433)

Pvu I (4683)

Sca I (4793)

Aat II (5235)

SnaB I (6009)

MCS (828-857) Bgl II* Xho I BamH I EcoR I Bgl II*

Pvu II (3243)

∆




Figure 5. pGAD424 map and MCS. Unique sites are in bold. pGAD424 (Bartel et al., 1993) is used togenerate a hybrid containing the GAL4 AD (a.a. 768–881) and another protein of interest (or a proteinencoded by a cDNA in a fusion library). The hybrid protein is expressed at low levels in yeast host cellsfrom a truncated ADH1 promoter and is targeted to the yeast nucleus by the SV40 T-antigen nuclearlocalization sequence (▲ ; Chien et al., 1991). pGAD424 contains the LEU2 gene for selection in Leu–

auxotrophic yeast strains.GenBank Accession: #U07647.


CCA AAC CCA AAA AAA GAG ATC GAA TTC CCG GGG ATC CGT CGA CCT GCA GAG ATC TAT GA

A TCG TAG ATA CTG AAA AAC CCC GCA AGT TCA CTT CAA CTG TGC ATC GTG CAC CAT CTC

Sma I BamH I Sal I Pst IEcoR I Bgl II

GAL4 AD

STOP (ORF 1)

STOP

(ORF 3)

STOP (ORF 2)


MATCHMAKER 5'AD LD-Insert- Screening Amplimer

812 •

755 •

871 •


2 µ ori

GAL4 AD

pGAD424 6.6 kb

PADH1

TADH1

LEU2

Col E1 ori

Ampr

Sph I (10)

Hind III (410)

Kpn I (484) Mlu I

(727)

Hind III (1105)

Sph I (1433)

EcoR V (1891)

Cla I (2487)

Bgl I (4442)

Pvu I (4692)

Sca I (4802)

Aat II (5244)

SnaB I (6018)

MCS (833-866) EcoR I

Sma I BamH I Sal I Pst I Bgl II

Pvu II (3253)

∆




Figure 6. pGAD GH map and MCS. Unique sites are in bold. pGAD GH (van Aelst et al., 1993) is usedto generate a hybrid containing the GAL4 AD (a.a. 768–881) and another protein of interest (or a proteinencoded by a cDNA in a fusion library). The hybrid protein is expressed at high levels in yeast host cellsfrom the full-length ADH1 promoter and is targeted to the yeast nucleus by the SV40 T-antigen nuclearlocalization sequence (▲ ; Chien et al., 1991). pGAD GH contains the LEU2 gene for selection in Leu–

auxotrophic yeast strains. pGAD GH was derived from pGAD GL (Figure 7) and is nearly identical to it,except in the promoter region. Also, an Sph I site (at bp 1600) in pGAD GL was mutagenized whenpGAD GH was constructed. Indicated bp's are approximate. No further pGAD GH sequence informationis available.


AAC CCA AAA AAA GAG ATC CTA GAA CTA GTG GAT CCC CCG GGC TGC AGG AAT TCG ATA TC

A AGC TTA TCG ATA CCG TCG ACC TCG AGG GGG GGC CCG GTA CCC AAT TCG CCC TAT AG

T GAG TCG TAT TAC AAT TCA CTG GCC GTC GTT TTA CAA CGT CGT GAC TGG GAA AAC CCT

GAT CTA TGA ATC GTA GAT ACT GAA AAA CCC CGC AAG TTC ACT TCA ACT GTG CAT CGT GCA

SK primer

KS primer

M13 –20 forward primer

T7 primer

STOP (ORF 3)

STOP (ORF 2)

STOP (ORF 1)

Hind III Xho I

BamH I

Sal I

Spe I Sma I Pst I EcoR I EcoR V

Cla I Apa I Kpn I

GAL4 AD GAL4 AD Sequencing Primer



2 µ ori

GAL4 AD

PADH1

TADH1

LEU2

Col E1 ori

Ampr

Sph I (6.9)

Hind III (0.4)

Hind III (1.2)

Aat II (5.5)

SnaB I (6.4)

Not I (3.45)

pGAD GH 7.8 kb

Sph I (0)

MCS (0.8–0.9) Spe I BamH I Sma I EcoR I Hind III Sal I Xho I Apa I

∆




Figure 7. pGAD GL map and MCS. Unique sites are in bold. pGAD GL (van Aelst et al., 1993) is usedto generate a hybrid containing the GAL4 AD (a.a. 768–881) and another protein of interest (or a proteinencoded by a cDNA in a fusion library). The hybrid protein is expressed at low levels in yeast host cellsfrom a truncated ADH1 promoter and is targeted to the yeast nucleus by the SV40 T-antigen nuclearlocalization sequence (▲ ; Chien et al., 1991). pGAD GL contains the LEU2 gene for selection in Leu–

auxotrophic yeast strains. Indicated bp's are approximate. No further pGAD GL sequence informationis available.


AAC CCA AAA AAA GAG ATC CTA GAA CTA GTG GAT CCC CCG GGC TGC AGG AAT TCG ATA TC

A AGC TTA TCG ATA CCG TCG ACC TCG AGG GGG GGC CCG GTA CCC AAT TCG CCC TAT AG

T GAG TCG TAT TAC AAT TCA CTG GCC GTC GTT TTA CAA CGT CGT GAC TGG GAA AAC CCT

GAT CTA TGA ATC GTA GAT ACT GAA AAA CCC CGC AAG TTC ACT TCA ACT GTG CAT CGT GCA

SK primer

KS primer

M13 –20 forward primer

T7 primer

STOP (ORF 3)

STOP (ORF 2)

STOP (ORF 1)

Hind III Xho I

BamH I

Sal I

Spe I Sma I Pst I EcoR I EcoR V

Cla I Apa I Kpn I

GAL4 AD GAL4 AD Sequencing Primer



2 µ ori

GAL4 AD

PADH1

TADH1

LEU2

Col E1 ori

Ampr

Sph I (0)

Hind III (0.4) Mlu I

(0.7)

Hind III (1.25)

Sph I (1.6)

EcoR V (2.0)

Cla I (2.6)

Bgl I (4.7)

Pvu I (5.0)

Sca I (5.1)

Aat II (5.5)

SnaB I (6.3)

∆

Not I (3.45)

pGAD GL 6.9 kb

MCS (0.8–0.9) Spe I BamH I Sma I EcoR I

EcoR V

Hind III Cla I Sal I Xho I Apa I Kpn I

Pvu II (3.4)

Pvu II (3.5)

Kpn I (0.5)



III. GAL4 DNA-Binding Domain (DNA-BD) Cloning Vectors

Figure 8. pAS2 map and MCS. Unique sites are in bold. pAS2 is a cloning vector used to generatefusions of a bait protein with the GAL4 DNA-BD (a.a. 1–147). pAS2 is synonomous with pAS1CHY2 (Harperet al., 1993) and carries the wild-type yeast CYHS2 gene, which confers sensitivity to cycloheximide intransformed yeast cells. The hybrid protein is expressed at high levels in yeast host cells from the full-length ADH1 promoter. The hybrid protein is targeted to the yeast nucleus by nuclear localizationsequences (Silver et al., 1984). pAS2 contains the TRP1 gene for selection in Trp– auxotropic yeaststrains. The Xba I site at bp 4763 (†) is methylation sensitive. The acidic amino acids of the HA epitopetag fused to the GAL4 DNA-BD causes high backgrounds in the two-hybrid assay when pAS2 is usedas a negative control. The HA epitope also leads to high backgrounds in some plasmid constructs basedon pAS2. pAS2 has been discontinued and replaced by its derivative pAS2-1 (Figure 9), which does notcontain an HA epitope.GenBank Accession: #U30496.

f1 ori

TRP1

CYH2

MCS (5981–6036)

Pst I Sal I

BamH I Sma I

Sfi I Nco I Nde I Nhe I

HA epitope

pAS2 8.4 kb

Col E1 ori

2 µ oriAmpr

PADH1

GAL4 BD

TADH1

Bgl II (3968)

EcoR I (3950)

EcoR I (5946)

EcoR V (4769)

Xba I† (4763)

EcoR V (3562)

Sac I (3297)

Nae I (2988)

EcoR V (2168)

Xba I (1969)

Xba I (982)

Hind III (3284)

Hind III (2397)

Hind III (5478)

Hind III (6246)

6094

•

GGT GGT CAT ATG GCC ATG GAG GCC CCG GGG ATC CGT CGA CCT GCA GCC AAG

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

Tyr Pro Tyr Asp Val Pro Asp Tyr Ala

CTA ATT CCG GGC GAA TTT CTT ATG ATT TAT GAT TTT TAT TAT TAAATAAGTT

Nde I Sal ISma I/ Xma I

Nco I

EcoR I

Pst I

pAS2-specific sequence

pAS2-specific sequence

HA epitope

Nhe I

BamH I

STOP (ORF 3)

STOP (ORF 2)

STOP (ORF 1)

5943

•

5991

•

6042

•

TCA TCG GAA GAG AGT AGT AAC AAA GGT CAA AGA CAG TTG ACT GTA TCG

5895

•

ATAAAAAAAATAAGTGTATACAAATTTTAAAGTGACTCTTAGGTTTTAAAACG

MATCHMAKER 5' BD Insert

Screening Amplimer & GAL4 DNA-BD Sequencing Primer

MATCHMAKER 3' BD Insert Screening Amplimer

GAL4 DNA-BD

Sfi I

a.a. 147



III. GAL4 DNA-Binding Domain (DNA-BD) Cloning Vectors continued

Figure 9. pAS2-1 map and MCS. Unique sites are in bold. pAS2-1 is a cloning vector used to generatefusions of a bait protein with the GAL4 DNA-BD (a.a. 1–147). pAS2-1 is derived from pAS2 (Figure 8)and hence from pAS1CHY2 (Harper et al., 1993) and carries the CYHS2 gene for cycloheximide sensitivity.The hybrid protein is expressed at high levels in yeast host cells from the full-length ADH1 promoter(PADH1). The hybrid protein is targeted to the yeast nucleus by nuclear localization sequences (Silver etal., 1984). The Xba I site at bp 4763 (†) is methylation sensitive. pAS2-1 contains the TRP1 gene forselection in Trp– auxotrophic yeast strains. Plasmid modification was performed at CLONTECH.GenBank Accession: #U30497.Compared with pAS2, pAS2-1 contains a neutral, short peptide instead of an HA epitope tag. Removingthe HA epitope tag and converting a.a.149 from Glu to Val completely eliminates the autonomousactivation activity of pAS2 (assayed in Y187 using the lacZ reporter; Holtz & Zhu, 1995). pAS2-1 has adifferent MCS than pAS2; however, the cloning sites that they have in common are in the same readingframe. Furthermore, the EcoR I, Xma I/Sma I, BamH I, Sal I, and Pst I sites in the pAS2-1 MCS are inthe same reading frames as those in pGBT9. Thus, inserts from pAS2 or pGBT9 can be excised andmoved to pAS2-1 without changing the reading frame. The EcoR I site in the pAS2-1 MCS is uniquebecause the EcoR I site in the CYHS2 gene in pAS2 was eliminated by site-directed mutagenesis.

STOP (ORF 3)

STOP (ORF 1)

5957

•

STOP (ORF 2)

Sal ISma I/ Xma I

Pst IBamH IEcoR I

6020

•

6088

•

pAS2-1 specific sequence

TCA TCG GAA GAG AGT AGT AAC AAA GGT CAA AGA CAG TTG ACT GTA TCG CCG GTA TTG CAA TAC CCA GCT TTG ACT CAT ATG GCC ATG GAG GCC GAA TTC CCG GGG ATC CGT CGA CCT GCA GCC AAG CTA ATT CCG GGC GAA TTT CTT ATG ATT TAT GAT TTT TAT TAT TAA ATAAGTTATAAAAAAAATAAGTG TATACAAATTTTAAAGTGACTCTTAGGTTTTAAAACG

MATCHMAKER 3' BD Insert Screening Amplimer

GAL4 DNA-BD

a.a. 147

5895

•

MATCHMAKER 5' BD Insert Screening Amplimer & GAL4 DNA-BD Sequencing Primer

Nde I Nco ISfi I

MCS (5969–6014)

Pst I Sal I

BamH I Sma I Xma I

EcoR I Sfi I

Nco I Nde I

pAS2-1 8.4 kb

Col E1 ori

f1 ori

2 µ oriAmpr

TRP1

CYH2

PADH1

GAL4 BD

TADH1

Bgl II (3969)

Xba I† (4763)

Sac I (3298)

Nae I (2989)

Xba I (1968)

Xba I (981)

Hind III (2397)

Hind III (3284)

Hind III (5477)

Hind III (6224)



III. GAL4 DNA-Binding Domain (DNA-BD) Cloning Vectors continued

Figure 10. pGBT9 map and MCS. Unique sites are in bold. pGBT9 (Bartel et al., 1993) is a cloning vectorused to generate fusions of a bait protein with the GAL4 DNA-BD (a.a. 1–147). The hybrid protein isexpressed at low levels in yeast host cells from a truncated ADH1 promoter. The hybrid protein istargeted to the yeast nucleus by nuclear localization sequences (Silver et al., 1984). pGBT9 contains theTRP1 gene for selection in Trp– auxotropic yeast strains.GenBank Accession: #U07646.

TCA TCG GAA GAG AGT AGT AAC AAA GGT CAA AGA CAG TTG ACT GTA TCG CCG GAA TTC CCG GGG ATC CGT CGA CCT GCA GCC AAG CTA ATT CCG GGC GAA TTT CTT ATG ATT TAT GAT TTT TAT TAT TAA ATA AGT TAT AAA AAA AAT AAG TGT ATA CAA ATT TTA AAG TGA CTC TTA GGT TTT AAA ACG

EcoR I Sma I

878 •

Pst ISal IBamH I

929 •

827 •

STOP (ORF 2)

GAL4 DNA-BD

STOP (ORF 1)

STOP (ORF 3)

MATCHMAKER 5' BD Insert Screening Amplimer &

GAL4 DNA-BD Sequencing Primer

983 •

MATCHMAKER 3'BD Insert

Screening Amplimer

pGBT9 5.5 kb

GAL4 BD

Ampr

PADH1 2 µ

ori

TADH1

TRP1

Col E1 ori

Hind III (410)

Hind III (1319)

Nae I (1115)

Pvu II (2118)

Xba I (1748)

Aat II (4109)

SnaB I (4883)

MCS (1004–1098) EcoR I Sma I BamH I Sal I Pst I



Figure 11. pCL1 map. pCL1 (Fields & Song, 1989) is a positive control plasmid that encodes the full-length, wild-type GAL4 protein. The GAL4 protein activates reporter genes under the control of aGAL4-responsive element (UAS

G). Thus, pCL1 is used as a positive control for the transcription assay

in GAL4-based MATCHMAKER Two-Hybrid Systems. This vector is a derivative of YCp50 referencedin Fields & Song, 1989. pCL1 has not been sequenced.

IV. Contol Plasmids

pCL1 15.3 kb

Ampr PADH1

GAL4

TADH1

LEU2

CEN4

Hind III

Hind III Hind III

pMB1 ori



Figure 12. pVA3 map. pVA3 (Iwabuchi et al., 1993) is a positive control plasmid that encodes a fusionof the murine p53 protein (a.a. 72–390) and the GAL4 DNA-BD (a.a. 1–147). The murine p53 cDNA(GenBank Accession #K01700) was cloned into pGBT9 (Figure 10). pVA3 has not been sequenced andit is not known whether any of the sites are unique.

Figure 13. pVA3-1 map. pVA3-1 is a positive control plasmid that encodes a fusion of the murine p53protein (a.a. 72–390) and the GAL4 DNA-BD (a.a. 1–147). The murine p53 cDNA (GenBank Accession#K01700) was cloned into pAS1CYH2 (a precursor of pAS2-1 [Figure 9]; Harper et al., 1993). The p53insert was derived from the plasmid described in Iwabuchi et al. (1993); plasmid modification wasperformed at CLONTECH. The Xba I site at bp 4763 (†) is methylation sensitive. pVA3-1 has not beensequenced and it is not known whether any of the sites are unique.

IV. Contol Plasmids continued

MCS Pst I Sal I BamH I EcoR I

pVA3-1 9.4 kb

Col E1 ori

f1 ori

2 µ oriAmpr

TRP1

CYH2

PADH1

GAL4 BD

TADH1

Bgl II (3969)

Xba I† (4763)

Sac I (3298)

Nae I (2989)

Xba I (1968)

Xba I (981)

Murine p53 insert

bp 371 (a.a. 72)

stop codon bp 1328

(a.a. 390)

bp 1345

Hind III (2397)

Hind III (3284)

Hind III (5477)

Hind III

pVA3 6.4 kb

Col E1 ori

2 µ ori

Amp r

TRP1

PADH1

TADH1

GAL4 BD

SnaB I

MCS EcoR I BamH I Sal I Pst I

Hind III

Nae I Hind III

Xba I

Pvu II

Aat II

Murine p53 insert

bp 371 (a.a. 72)

stop codon

bp 1345

bp 1328 (a.a. 390)



BamH I Hind III

Pvu I

Pvu I

Xba I, Pvu I

Xba I

Xba I*

EcoR I

EcoR I

EcoR I

Hind III

Sph I

Sph I

Sal I

Sph ISal I

pTD1 ~15 kb

Ampr

PADH1

2 µ ori

GAL4 AD

TADH1

LEU2

pBR322 ori

∆

Pvu II

SV40 large T-antigen

bp 4559 (a.a. 87)

Hind III bp 4002

Nde I bp 3808

Hind III bp 3477

bp 2539

Stop codon bp 2693 (a.a. 708)

Pvu II bp 3509


Figure 15. pTD1-1 map. pTD1-1 is a positive control plasmid that encodes a fusion of the SV40 largeT-antigen (a.a. 87–708) and the GAL4 AD (a.a. 768–881). The SV40 large T-antigen cDNA (GenBankLocus SV4CG) was cloned into pACT2 (Figure 3). The SV40 T-antigen insert was derived from theplasmid referenced in Li & Fields (1993); plasmid modification was performed at CLONTECH. pTD1-1has not been sequenced and it is not known whether any of the sites are unique.

Figure 14. pTD1 map. pTD1 (Li & Fields, 1993) is a positive control plasmid that encodes a fusion ofthe SV40 large T-antigen (a.a. 86–708) and the GAL4 AD (a.a. 768–881). The SV40 large T-antigencDNA was cloned into pGAD3F (Chien et al., 1991). pTD1 has not been sequenced and it is not knownwhether any of the sites are unique.

pTD1-1 10.1 kb

MCS

Bgl II HA epitope

Nde I Sfi I

Nco I Sma I Xma I

BamH I EcoR I Xho I Bgl II

Not I (4163)

Cla I (2626)

Hpa I (1581)

Xba I (598)

2 µ ori

GAL4 AD

PADH1

TADH1

LEU2

Col E1 ori

Ampr

Not I (4345)

loxP

HA

SV40 large T-antigen

bp 4559 (a.a. 87)

Hind III bp 4002

Nde I

bp 3808

Hind III bp 3477

Stop codon bp 2693 (a.a. 708)

bp 2539

Hind III (4739)

Hind III

♦

∆



Figure 16. pLAM 5' map. pLAM5' is a false-positive detection plasmid that encodes a fusion of thehuman lamin C protein (a.a. 66–230) and the GAL4 DNA-BD (a.a. 1–147). The lamin C cDNA insert(GenBank Accession #M13451) was derived from the plasmid referenced in Bartel et al. (1993), and wascloned into pGBT9 (Figure 10). Plasmid modification was performed at CLONTECH. pLam5' has notbeen sequenced and it is not known whether any of the sites are unique.

Figure 17. pLAM 5'-1 map. pLAM5'-1 is a false-positive detection plasmid that encodes a fusion of thehuman lamin C protein (a.a. 66–230) and the GAL4 DNA-BD (a.a. 1–147). The lamin C cDNA insert(GenBank Accession #M13451) was derived from the plasmid referenced in Bartel et al. (1993), and wascloned into pAS2-1 (Figure 9). Plasmid modification was performed at CLONTECH.pLAM5'-1 has not been sequenced and it is not known whether any of the sites are unique.


pLAM5' 6.0 kb

Col E1 ori

2 µ ori

Amp r

TRP1

PADH1

TADH1

GAL4 BD

SnaB IMCS EcoR I Sma I BamH I Sal I Pst I

Hind III

Nae I

Hind III

Xba I

Pvu II

Aat II

Human lamin C

bp 330 (a.a. 66)

bp 825 (a.a. 230)

Hind III bp 645

MCS Pst I Sal I BamH I Sma I EcoR I Sfi I Nco I Nde I

pLAM 5'-1 9.1 kb

Col E1 ori

f1 ori

2 µ oriAmpr

TRP1

CYH2

PADH1

GAL4 BD

TADH1

Bgl II (3969)

Xba I† (4763)

Sac I (3298)

Nae I (2989)

Xba I (1968)

Xba I (981)

Human lamin C

bp 330 (a.a. 66)

bp 825 (a.a. 230)

Hind III (5477)

Hind III

Hind III (2397)

Hind III (3284)

Hind III bp 645



V. References

Bartel, P. L., Chien, C.-T., Sternglanz, R. & Fields, S. (1993) Using the two-hybrid system to detect protein-protein interactions.In Cellular Interactions in Development: A Practical Approach., ed. Hartley, D.A. (Oxford University Press, Oxford) pp. 153–179.

Chien, C. T., Bartel, P. L., Sternglanz, R. & Fields, S. (1991) The two-hybrid system: A method to identify and clone genes forproteins that interact with a protein of interest. Proc. Nat. Acad. Sci. USA 88:9578–9582.

Durfee, T., Becherer, K., Chen, P. L., Yeh, S. H., Yang, Y., Kilbburn, A. E., Lee, W. H. & Elledge, S. J. (1993) The retinoblastomaprotein associates with the protein phosphatase type 1 catalytic subunit. Genes Devel. 7:555–569.

Fields, S. & Song, O. (1989) A novel genetic system to detect protein-protein interactions. Nature 340:245–247.

Harper, J. W., Adami, G. R., Wei, N., Keyomarsi, K. & Elledge, S. J. (1993) The p21 Cdk-interacting protein Cip1 is a potentinhibitor of G1 cyclin-dependent kinases. Cell 75:805–816.

Iwabuchi, K., Li, B., Bartel, P. & Fields, S. (1993) Use of the two-hybrid system to identify the domain of p53 involved inoligomerization. Oncogene 8:1693–1696.

Li, B. & Fields, S. (1993) Identification of mutations in p53 that affect its binding to SV40 T antigen by using the yeast two-hybridsystem. FASEB J. 7:957–963.

Li, L., Elledge, S. J., Peterson, C.A., Bales, E. S. & Legerski, R. J. (1994) Specific association between the human DNA repairproteins XPA and ERCC1. Proc. Natl. Acad. Sci. USA 91:5012–5016.

Silver, P. A., Keegan, L. P. & Ptashne, M. (1984) Amino terminus of the yeast GAL4 gene product is sufficient for nuclearlocalization. Prod. Natl. Acad. Sci. USA 81:5951–5955.

van Aelst, L., Barr, M., Marcus, S., Polverino, A. & Wigler, M. (1993) Complex formation between RAS and RAF and other proteinkinases. Proc. Natl. Acad. Sci. USA 90:6213–6217.



VI. Plasmid and System Ordering Information

Product Size Cat.#

MATCHMAKER Two-Hybrid System each K1605-1

MATCHMAKER Two-Hybrid System 2 each K1604-1

Two-Hybrid cDNA Library Construction Kit each K1607-1

MATCHMAKER cDNA Libraries many

MATCHMAKER DNA-BD Insert Screening Amplimer Set 100 rxn 5417-1

MATCHMAKER AD LD-Insert Screening Amplimer Set 100 rxn 9103-1

GAL4 Activation Domain Sequencing Primer 2.5 µg 6473-1

GAL4 Binding Domain Sequencing Primer 2.5 µg 6474-1

GAL4 AD mAb 20 µg 5398-1

GAL4 DNA-BD mAb 20 µg 5399-1

Vectors available separately

pACT2 40 µg K1604-A

pGAD10 40 µg 6180-1

pGAD424 40 µg K1605-B

pGAD GH 40 µg 6182-1

pGAD GL 40 µg 6181-1

pAS2-1 40 µg K1604-B

pGBT9 40 µg K1605-A



APPENDIX. Additional MATCHMAKER Plasmid Information

TA

BLE

III.

MA

TC

HM

AK

ER

GA

L4

TW

O-H

YB

RID

SY

ST

EM C

LON

ING

VE

CT

OR

S

Sel

ectio

n on

Siz

eD

iagn

ostic

Gen

Ban

kV

ecto

rS

yste

ma

Des

crip

tion

SD

Med

ium

(kb)

R.E

. Site

s (k

b)A

cces

sion

#R

efer

ence

s

pAC

TM

M L

ibra

ries

GA

L4 A

D,

–Leu

7.65

Eco

R I

not a

vaila

ble

Dur

fee

et a

l., 1

993;

LEU

2, A

mpr ,

(3.0

, 3.0

5, 1

.6)

Elle

dge,

per

s. c

omm

.H

A e

pito

pe ta

g

pAC

T2

GA

L4 2

H-2

GA

L4 A

D,

–Leu

8.1

Hin

d III

U29

899

Li e

t al.,

199

4;(#

K16

04-1

) &

LEU

2, A

mpr ,

(7.3

, 0.8

)E

lledg

e, p

ers.

com

m.

MM

Lib

rarie

sH

A e

pito

pe ta

g

pAS

2(#

K16

05-D

)bG

AL4

DN

A-B

D,

–Trp

8.4

Hin

d III

U30

496

Dur

fee

et a

l., 1

993;

TR

P1,

Am

pr(4

.6, 2

.2, 0

.9, 0

.7)d

Har

per

et a

l., 1

993

pAS

2-1

cG

AL4

2H

-2G

AL4

DN

A-B

D,

–Trp

8.4

Hin

d III

U30

497

Har

per

et a

l., 1

993

(#K

1604

-1)

TR

P1,

Am

pr , C

YH

s 2(4

.6, 2

.2, 0

.9, 0

.7)d

pGA

D10

MM

Lib

rarie

sG

AL4

AD

,–L

eu6.

6H

ind

IIIU

1318

8B

arte

l et a

l., 1

993

LEU

2, A

mpr

(5.9

, 0.7

)e

pGA

D42

4G

AL4

2H

GA

L4 A

D,

–Leu

6.6

Hin

d III

U07

647

Bar

tel e

t al.,

199

3(#

K16

05-1

)LE

U2,

Am

pr(5

.9, 0

.7)e

pGA

D G

HM

M L

ibra

ries

GA

L4 A

D,

–Leu

7.9

Hin

d III

not a

vaila

ble

van

Ael

st e

t al .,

199

3LE

U2,

Am

pr(7

.1, 0

.5, 0

.3)

pGA

D G

LM

M L

ibra

ryG

AL4

AD

,–L

eu6.

9H

ind

IIIno

t ava

ilabl

eva

n A

elst

et a

l ., 1

993

LEU

2, A

mpr

(6.1

, 0.5

, 0.3

)

pGB

T9

GA

L4 2

HG

AL4

DN

A-B

D,

–Trp

5.5

Hin

d III

U07

646

Bar

tel e

t al.,

199

3(#

K16

05-1

)T

RP

1, A

mpr

(4.6

, 0.9

)

aK

ey t

o sy

stem

abb

revi

atio

ns:

GA

L4 2

H-2

= M

AT

CH

MA

KE

R T

wo-

Hyb

rid S

yste

m 2

(#K

1604

-1);

GA

L4 2

H =

MA

TC

HM

AK

ER

Tw

o-H

ybrid

Sys

tem

(#K

1605

-1).

Som

e pl

asm

ids

are

also

avai

labl

e se

para

tely

(se

e S

ectio

n V

I).

bT

he M

AT

CH

MA

KE

R S

uppl

emen

t Kit

(#K

1605

-D)

was

rep

lace

d by

the

MA

TC

HM

AK

ER

Tw

o-H

ybrid

Sys

tem

2 (

#K16

04-1

).c

pAS

2-1

is a

der

ivat

ive

of th

e pl

asm

id d

escr

ibed

in th

is r

efer

ence

; the

pla

smid

was

mod

ified

at C

LON

TE

CH

.d

The

Eco

R I

site

is u

niqu

e in

pA

S2-

1, b

ut n

ot in

pA

S2.

epG

AD

424

is li

near

ized

by

dige

stio

n w

ith S

al I;

pG

AD

10 d

oes

not c

onta

in a

Sal

I si

te.



APPENDIX. Additional MATCHMAKER Plasmid Information continued

TA

BLE

IV.

MA

TC

HM

AK

ER

GA

L4

TW

O-H

YB

RID

SY

ST

EM

CO

NT

RO

L P

LAS

MID

S

Sel

ectio

n on

Siz

eV

ecto

raS

yste

mD

escr

iptio

nS

D M

ediu

m(k

b)D

iagn

ostic

R.E

. Site

s (k

b)R

efer

ence

s

pCL1

GA

L4 2

H &

2H

-2w

ild-t

ype

full-

leng

th G

AL4

–Leu

~15

.3H

ind

IIIF

ield

s &

Son

g, 1

989

(#K

1605

-1 &

gene

in a

YC

p50

deriv

ativ

e,(~

11.2

, 2.8

, 1.8

)#K

1604

-1)

LEU

2 , A

mpr

pLA

M5'

GA

L4 2

HH

uman

lam

in C

(66–

230)

–Trp

6.0

Hin

d III

Bar

tel e

t al.,

199

3(#

K16

05-1

)in

pG

BT

9, T

RP

1, A

mpr

(4.6

, 0.8

, 0.6

)

pLA

M5'

-1G

AL4

2H

-2H

uman

lam

in C

(66–

230)

–Trp

~9.

0H

ind

IIIB

arte

l et a

l., 1

993

(#K

1604

-1)

in p

AS

2-1

TR

P1,

Am

pr(4

.6, 2

.2, 0

.9, 0

.85,

0.4

)

pTD

1G

AL4

2H

SV

40 la

rge

T-a

ntig

en (8

4–70

8)–L

eu~

15.0

Hin

d III

Li &

Fie

lds,

199

3;(#

K16

05-1

)in

pG

AD

3F, L

EU

2, A

mpr

(12,

1.3

, 1.2

, 0.5

)C

hien

et a

l., 1

991

pTD

1-1

GA

L4 2

H-2

SV

40 la

rge

T-a

ntig

en (8

4–70

8)–L

eu~

10.0

Hin

d III

Li &

Fie

lds,

199

3;(#

K16

04-1

)in

pA

CT

2, L

EU

2, A

mpr

(7.3

, 1.2

, 1.0

, 0.5

)

pVA

3G

AL4

2H

mur

ine

p53

(72–

390)

in p

GB

T9

–Trp

6.4

Hin

d III

Iwab

uchi

et a

l., 1

993

(#K

1605

-1)

TR

P1 ,

Am

pr(4

.6, 1

.8)

pVA

3-1

GA

L4 2

H-2

mur

ine

p53

(72–

390)

in p

AS

2-1

–Trp

9.4

Hin

d III

Iwab

uchi

et a

l., 1

993

(#K

1604

-1)

TR

P1 ,

Am

pr(4

.6, 2

.2, 1

.7, 0

.9)

Chi

en e

t al.,

199

1

apL

AM

5', p

LAM

5'-1

, pT

D1-

1, a

nd p

VA

3-1

are

deriv

ativ

es o

f the

pla

smid

s de

scrib

ed in

the

indi

cate

d re

fere

nces

; pla

smid

s w

ere

mod

ified

at C

LON

TE

CH

.



Notice to PurchaserPractice of the two-hybrid system is covered by US Patents #5,283,173 and #5,468,614 assigned to the Research Foundationof the State University of New York. Purchase of any CLONTECH two-hybrid reagent does not imply or convey a license topractice the two-hybrid system covered by these patents. Commercial entities purchasing these reagents must obtain a licensefrom the Research Foundation of the State University of New York before using them. CLONTECH is required by its licensingagreement to submit a report of all purchasers of two-hybrid reagents to SUNY Stony Brook. Please contact Carol Dempster,Ph.D., at the Long Island Research Institute for license information (Tel: 516-361-6800; Fax: 516-361-6840).

All plasmids (except for pACT2 and pAS2-1) are licensed from The Research Foundation of the State University of New York.pACT2 and pAS2-1 are licensed from Baylor University. CLONTECH encourages researchers not to redistribute the plasmidsor yeast strains without prior written consent.

1997, CLONTECH Laboratories, Inc. All rights reserved.

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