M13 vectors with 17 polymerase promoters: transcription limited by oligonodeotides

Synthetic sequences corresponding to T7 promoters have been cloned into the vectors H13mplO and M13mpl8 at the EcoRl site and into M13mpll and M13mpl9 at the Hlndlll site, creating mICE 10,11,18 and 19. These sequences of 33-base pairs include the region essential for activity of T7 promoters (1). The inserts preserve the g-galactosldase cloning marker and restore the initial unique restriction enzyme cleavage site. The homologous region of an SP6 promoter (2) was cloned likewise, but found to be far less efficient per unit of homologous RNA polymerase. TheBe vectors can be used Juet as the H13mp vectors (3) for cloning, sequencing and in vitro mutagenesis, but it is not necessary to redone in order to transcribe the genes in vitro. In all these constructions, the promoter has been inserted on the opposite side of the polylinker from the universal DNA priming site, and thus directs RNA synthesis from 5 bases upstream of the polylinker across the cloned genes in the opposite sense to primed DNA synthesis upon the single-stranded DNA. This leads to three major advantages: 1. Single-stranded probes can be prepared from an insert in either sense. The RNA transcript will be in the viral DNA sense and, as in all vectors producing single-stranded DNA, DNA probes produced by priming will be in the complementary sense. 2. Sequences can be determined readily from both ends of a cloned gene: using 3'-deoxyribonucleoside trlphosphates and T7 RNA polymerase CO for sequences from the 5' end in the viral DNA sense, and using the methods of Sanger et al • (5) for sequences of the opposite strand. 3. The 3' end of a transcript can be defined with an oligonucleotide, which is more versatile than using restriction enzyme cleavage. Single-stranded viral DNA is used as a template for DNA synthesis from an oligonucleotide-either the universal primer or within the target gene. After the new strand has crossed the promoter (we allow it 5 min at 37°C), transcription is initiated by addition of rNTPs and T7 polymerase, and ends at the site defined by the primer. Furthermore, it ia facile to generate a series of 3'-truncated RNAs with different oligonucleotide primers (without the laborious process of generating deletions and recloning the products), for use in studies of RNA or protein functions or to map antigenlc determinants. The mICE vectors will be Bade available by Amersham International PLC.