A convenient cloning vector containing the GAL4 DNA-binding domain

Getw, 118 (1992) 14sI44

0 1992 Elwvier

GENE

065X5

Brief

Notes

Science Publishers

B.V. All rights reserved.

A convenient

cloning vector containing

(Recombinant

fusion proteins;

DNA;

Loretta Raycroft

Reccivcd

27 January

the GAL4 DNA-binding

domain

trans-activation)

and Guillermina

by R. Padmanabhan:

143

0378-I 119~92~905.00

Lozano

1992: Acccptcd:

I I March 1992: Received at publishers:

4 May 1992

SUMMARY

A DNA fragment encoding the yeast GAL4 DNA-binding domain (amino acids 3-147) was cloned into a convenient vector. This vector contains unique restriction sites at both the 5’ and 3’ ends and allows the generation of fusion proteins containing the GAL4 DNA-binding domain. These fusion proteins can be tested for their ability to activate transcription.

The function of the yeast transcription factor GAL4 has been studied in detail. Functionally, GAL4 consists of a DNA-binding domain (aa 1-147) and a transactivation domain (aa 148-881) (Keegan et al., 1986). Expression of GAL4 in mammalian cells will specifically transactivate a target gene containing the UAS to which GAL4 binds (Kakidani and Ptashnc, 1988). The DNA-binding domain of GAL4 has been extremely useful in the analysis of potential transcription factors in tissue culture. In particular, fusion proteins of the GAL4 DNA-binding domain and Myb or ElA have been important in defining transactivation domains (Weston and Bishop, 1989; Lillie and Green, 1989). Currently available plasmids encoding the GAL4 DNA-

C‘orre.vpor,tkrrc,r IO; Dr. G. Lozano, The University

Blvd., Box I I, Houston. Fax (713) 794-4295 Abbreviations: scription

factor:

Department

of Texas. M.D. Anderson

aa. amino

of Molecular

Cancer Center,

Genetics.

1515 Holcombe

TX 77030. USA. Tel. (713) 792-8945;

acid(s):

bp, base pair(s);

GAL4, gent encoding

GAL4:

G.4L4,

kb, kilobase

yeast tranor 1000 bp;

nt, nucleotidc(s); MCS, multlplc cloning site(s); PolIk, Klenow (large) fragment of E. co/i DNA polymerase I: UAS. upstream activating scqucnce.

binding domain allow the generation of fusion proteins in which GAL4 is placed only at the N terminus (Sadowski et al., 1988; 1992). Since the tumor suppressor ~53 contains a putative DNA-binding domain at the C terminus, we wished to generate a vector that would allow us to insert the GAL4 DNA-binding domain at the C-end of ~53. To do this, we digested pLK8. a yeast cloning vector containing the entire GAL4 sequence (a gift from P. Silver), with AccI, which cuts 55 bp upstream from the GAL4 start codon, and treated it with BAL 31 followed by Pollk. Subsequent digestion with CkrI, which cuts DNA at the site corresponding to aa 147 of GAL4, was used to clone this fragment into a pBluescript SK(-) plasmid (Stratagcne, La Jolla, CA) cut with Sinai + ClrlI. The resulting plasmid. pLR310, contained two TAG stop codons in the 5’ linker in frame with the GAL4 aa sequence. We linearized pLR3 10 with BarnHI, blunt-ended it with PolIk, and religated it to generate pLR60 (Fig. 1). This procedure shifted the two stop codons to a different reading frame and generated a new ClcrI site at the 5’ end of GAL4 which is sensitive to dutn methylation and is not cleaved by C/t/I. The nt sequences of both 5’ and 3’ ends of this GAL4 insert are shown in Fig. 1B. Since MCS are present at both ends of GAL4, this plasmid can bc used to gcncrate inframe fusion proteins to the GAL4 DNA-binding domain,

B 5' end T3 primer ATT

AAC

CCT

CAC

SacI

TAA

AGA

ACT

4

AGT

Not1

AGG GAA CAA AAG CTG GAG CTC CAC CGC GGT GGC GGC CGC

SpeI TCT

Sac11

5

6

7

8

9

10

11

12

GGA TCG ATC CCC CTG TCT TCT ATC GAA CAA GCA TGC GAT

XbaI

SphI

3' end xinc11 AccI

ApaI DraII

SalI

ACT GTA TCG ATA CCG TCG ACC TCG AGG GGG GGC CCG GTA CCC AAT TCG CCC ClaI

XhoI

XprrI

TAT AGT GAG TCG TAT T T7 primer Fig.

I. Maps of pLR60 and GAL4. (A) Some of the restriction

arrow indicates

the 5’.to-3’

direction

of the GAL4 sequcnccs.

sites in the MCS and the location

of T3 and T7 promoters

(boxes) arc shown. The long

The mtcrnal XhoI site in GAL4 occurs at aa 73. The C&I site marks the end of GAL4 (an

117). The C/u1 site in parentheses is sensitive to &rn mcthylation. (B) Scquencc of 5’ and 3’ ends of GAL4 (see A). Scquencc was determined usmg the didcoxy chain-termination method. T7 or T3 primers. and Scquenasc (USB, Cleveland. OH). Codons arc drawn in frame with the G11L-I acqucncc.

Numbers primers

above the nt sequcncc

refer to GALJ-encoding

aa [see Laughon

and Gestcland

(19X4) for GnL4

scqucncc].

Restriction

sites and T3 and T7

arc underlined.

in the middle, or at the C terminus of another protein. Plasmid pLR60 has been used successfully to generate p53GAL4 fusion proteins and to test the function of p53 as a transcription factor (Raycroft et al., 1990; 1991). Support

was provided

Laughon.

A. and Gcstcland.

c~ere~i.~icrr GAL4

gent.

Lillic, J.W. and Green, Ela protein. Raycroft,

Raycroft.

L.. Wu H. and Lozano,

L.. Schmidt.

Sadou-ski, in mam-

Keegan, L.. Gill, G. and Ptaahne, M.: Separation of DN.4 binding from the transcription-activating function of a cukaryotic regulator> protcin. Science 23 1 (1986) 699-704.

Sadowski,

activation

b> the adenovti-us

G.: Transcriptional

mutants

acti\sation

b) wild-

of the p53 anti-oncogenc.

J.R.. Yeas. K.. Hao, M. and LoLano, for transcriptional

acitivity.

Scicncc G:

Ana-

Mol. Cell. Biol.

II

1.. Bell. B.. Broad, P. and Hollis, M.: G,4L4 fusion vectors fat

expression gent expression

of the S~c.c~hrrw~iir~c~c~~

338 (1989) 39-44.

)sis of p53 mutants (1991) 6067-6074. REFERENCES

structure

M.R.: Transcription

Nature

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Kakidani. H. and Ptashne. M.: GAL4 activates malian cells. Cell 52 (1988) 161-167.

R.F. Primnr)

Mol. Cell. Biol. 4 (1984) 260-267.

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cells. Gcnc

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