Figure 2 . Polymerisation of a 42 bp `minimal' RRE sequence. Oligonucleotides 5'-TCGACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCC-3' and 5'-TCGAGGCCTGTACCGTCAGCGTCATTGACGCTGCGCCCATAG-3' were synthesised on an Applied Biosystem 380A. They were purified on Nensorb columns (DuPont) and annealed in 100 mM NaCl. The resulting 42 bp fragment was cloned between the Sal I and Xho I sites of pBluescript (Stratagene). A Pvu II/ Hin dIII fragment from pGFIB1-phe was then cloned in the 1X vector cut by Sma I and Hin dIII, generating the 1X+S vector. A Xba I- Sal I fragment from this vector (containing one copy of the RRE sequence and the suppressor tRNA gene) was then cloned into a Xba I/ Xho I cut 1X vector (containing one copy of the RRE sequence). The resulting 2X+S vector was then used as a source of a Xba I- Sal I fragment for the next round of cloning in the 1X vector to generate the 3X+S vector. Iterative cloning led to vectors containing one to six tandem copies of the minimal RRE sequence. These vectors were cut with Sal I and Xho I and the DNA was separated on a 8-25% acrylamide gradient gel (Phastgel, Pharmacia). The gel was then silver stained. Lane 1 , 123 bp size marker (Pharmacia); lane 2, RRE 1X (42 bp); lane 3, RRE 2X (84 bp); lane 4, RRE 3X (126 bp); lane 5, RRE 6X (252 bp).

A 42 bp core sequence of the RRE has been defined as the minimal element required to bind HIV-1 regulatory protein Rev ( 2 ). In order to study the binding of Rev with this target sequence, we constructed a family of vectors harbouring one to six copies of this 42 bp core RRE sequence. This was easily achieved by cloning the 42 bp synthetic sequence between the Sal I and Xho I sites of pBluescript, and adding the tRNA _CUA Phe gene from pGFIB1-phe ( 3 ) next to it between the Xba I and the Hin dIII sites. The successive nX+S fragments were then obtained with Xba I/ Xho I restriction cuts and cloned into the Xba I/ Sal I sites of vector 1X (Fig. 2 ).

One major advantage of this polymerisation method is that it is sequence and size independent. It can therefore be used to obtain short polymeric sequences derived from bacterial operator sequences or eucaryotic UAS for example. This can be very useful for DNA binding protein purification or band-shift assays. The orientation of the copies can be predefined in order to obtain direct or inverted repeats. The DNA tag, a suppressor tRNA gene cassette, is large enough to be easily detected by standard restriction analysis of the constructs, but its small size does not overload the vector. Although the insert gets harder to clone as the polymerised sequence increases in size, the suppression provides a very effective selection for the correct constructs in strains with an adequate genetic background.

Two or more different sequences can be combined in this protocol to build a more complex repetitive motif. This enables to assemble a gene coding for a protein where for example several copies of a repeated peptide motif are linked by a spacer sequence. Moreover, the suppressor tRNA gene has been successfully used as a DNA tag for other difficult cloning experiments.

REFERENCES

2 Kjems,J., Brown,M., Chang,D.D. and Sharp,P.A. (1992) Proc. Natl. Acad. Sci. USA, 88, 683-687. MEDLINE Abstract

3 Masson,J.M. and Miller,J.H. (1986) Gene, 47, 179-183. MEDLINE Abstract

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