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Nucleic Acids Research, 1990, Vol. 18, No. 17 5173-5180
© 1990

MOLECULAR BIOLOGY

A novel selection system for recombinational and mutational events within an intron of a eucaryotic gene

Thomas Porter, Sandra L. Pennington*, Gerald M. Adair1, Rodney S. Nairn1 and John H. Wilson*

Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine Houston, Texas 77030, USA 1The University of Texas M.D. Anderson Cancer Center Science Park-Research Division, Smithville, TX 78957, USA

*To whom correspondence should be addressed

Received May 15, 1990. Revised July 31, 1990. Accepted July 31, 1990.

In order to identify a poison sequence that might be useful in studying illegitimate recombination of mammalian cell chromosomes, several DNA segments were tested for their ability to interfere with gene expression when placed in an intron. A tRNA gene and its flanking sequences (267 bp) were shown to inhibit SV40 plaque formation 100-fold, when inserted into the intron in the T-antigen gene. Similarly, when the same DNA segment was placed in the second intron of the adenosine phosphoribosyl transferase (APRT) gene from CHO cells, it inhibited transformation of APRT CHO cells 500-fold. These two tests indicated that the 267-bp DNA segment contained a poison sequence. The poison sequence did not affect replication since the replication of poisoned SV4O genomes was complemented by viable SV40 genomes and poisoned APRT genes were stably integrated into cell chromosomes. Cleavage of the poison sequence in the SV40 T-antigen intron by restriction enzymes indicated that the tRNA structural sequences and the 5' flanking sequences were not required for inhibition of SV40 plaque formation. Sequence analysis of viable mutant SV40, which arose after transfection of poisoned genomes, localized the poison sequence to a 35 bp segment immediately 3' of the tRNA structural sequences.


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