Fig. 4.

Increasing translation efficiency of connexins in cell-free translation systems. α₁Cx43 and β₁Cx32 cDNAs were cloned into standard transcription vectors (lanes 1, 3), and into the transcription vector pSP64T (lanes 2, 4) that contains the 5′ noncoding region of Xenopus β-globin as a translation enhancer upstream of the cDNA. cDNAs were transcribed and translated in rabbit reticulocyte lysates in the absence of microsomes. In each lane 0.5 μL of translation reaction was analyzed on SDS-polyacrylamide gels and translation products were visualized by autoradiogra- phy. Note the dramatically increased translation efficiency of transcripts containing the translation enhancer sequence (lanes 2, 4 vs 1, 3). Additional bands detectable on the gels represent aggregates of translation products, and translation products initiated at AUG codons located downstream from the connexin authentic initiation codons, and were generated mostly due to nonoptimized conditions. Gels were purposely overexposed to visualize these products. (Compare Fig. 5 for connexin-expression profiles obtained after optimizing translation conditions.) Substantially increasing the translation efficiency of connexins allowed the subsequent synthesis and assembly of oligomeric connexons in cell-free translation systems, and to analyze the assembly behavior of different connexin isotypes.