Membrane insertion of gap junction connexins: polytopic channel forming membrane proteins

For the in vitro transcription of efficiently translated synthetic connexin RNAs, rat cq, human/~1, rat/~2, rat/33, and rat a3 GJ cDNAs were cloned into the BgUI site of the transcription vector pSP64T (Krieg and Melton, 1984) . The general method used for the construction involved mutation of a region rvl0-30 bases upstream of the initiation codon to produce a BamHI site. Either the BamHI site in the polylinker or a BgiII linker ligated to restriction enzyme-cut DNA was used to generate BamH -BamHI or BamHI-BglII fragments of the GJ cDNAs. Specifically, the fll GJ construct contains the nucleotides 51-996 of the human/31 GJ cDNA with a BgiII linker ligated into the StuI site 82 bp downstream from the stop codon of the/31 GJ eDNA. The/32 GJ construct contains nucleotides 259 to ~,2350 of the rat/32 GJ cDNA. The/$3 GJ construct contains nucleotides 71-895 of the rat ~3 sequence. The cq GJ construct contains nucleotides 191-1668 of the t~l GJ cDNA with a BgilI linker ligated into the AflII site, after filling in the protruding ends with Klenow DNA polymerase. The ct3 GJ construct contains nucleotides 201-1705 of the rat ct3 GJ eDNA. Resulting constructs consisted of the connexin coding region and ,,010--30 upstream bases and 100-300 downstream bases of the GJ cDNAs, flanked by the 5' and 3' noncoding regions of Xenopus/~-globin eDNA.