XhoI

Abstract: The coding region of the bacterial chloramphenicol acetyltransferase (CAT) gene is wide1y used as an indicator gene in gene transfer experiments dealing with regulation of transcription in eukaryotes. Chimaeric CAT fusion genes are especially useful because no endogenous CAT activity is present in eukaryotic ce11s and because CAT enzyme activity can be monitored by a rapid and sensitive assay (1). In order to simplify the construction of hybrid CAT genes, we have constructed the plasmids pBLCA T2 and pBLCAT3. The coding region of the CA T gene as well as the small t intron and polyadenylation signals from SV40 were inserted into the polylinker region of the high copy number plasmid pUC18 (2). Unique BglII and XhoI restrietion sites were introduced upstream of the CAT coding region by insertion of synthetic linkers. A BamHI site at the 3' end of the transcription unit was converted into adam methylation sensitive ClaI site by partial digestion with BamHI, filling in and re -ligation. In the promoterless construction pBLCAT3 eight unique restriction sites are suitable for insertion of different eukaryotic promoters at the 5' end of the CAT gene. Four additional unique restriction sites rnake the insertion of regulatory signals 3' of the CAT gene possible and enable the excision of the intact fusion gene from the prokaryotic vector. The presence of the Herpes simplex virus tk promoter in pBLCAT2 permits the analysis of the effects of putative regulatory elements on a heterologous eukaryotic promoter. A BamHIIBgllI fragment from the HSV tk linker scanning mutant LS 115/ 105 (3) spanning the promoter from 105 to + 51 was inserted into the corresponding restriction sites of pBLCAT3 thereby generating pBLCAT2. The modified polylinker regions at the 5' and the 3' ends have been sequenced and compiled sequences for both plasmids are available on request.

Abstract: This paper reports a cleavage site map of Tn5 for restriction enzymes BamHI, Bg/I, Bg/II, Hind II, HindIII, HpaI, Sa/I, Aval, SmaI, XhoI, PstI, PvuII, HaeII and HaeIII that was determined by the analysis of restriction enzyme cleavage patterns of ColEl, two independent ColEl::Tn5 plasmids, and a ColEl::Tn5 deletion derivative. Ba/I, EcoRI, KpnI, and PvuI do not cleave Tn5. Construction and analysis of in vitro-generated deletions of a ColEl::Tn5 plasmid limit the sequences encoding neomycin resistance to a 1500-base-pair-long segment of Tn5. Insertion of DNA at a Bg/II site within this segment results in loss of the neomycin resistance phenotype. Since this Bg/II site lies in an inverted repeat region, sequences within this repeat seem to be involved in the expression of neomycin resistance.