Hiroshi Kanazawa

We developed mobility shift analysis of single-stranded DNAs on neutral polyacrylamide gel electrophoresis to detect DNA polymorphisms. This method follows digestion of genomic DNA with restriction endonucleases, denaturation in alkaline solution, and electrophoresis on a neutral polyacrylamide gel. After transfer to a nylon membrane, the mobility shift due to a nucleotide substitution of a single-stranded DNA fragment could be detected by hybridization with a nick-translated DNA fragment or more clearly with RNA copies synthesized on each strand of the DNA fragment as probes. As the mobility shift caused by nucleotide substitutions might be due to a conformational change of single-stranded DNAs, we designate the features of single-stranded DNAs as single-strand conformation polymorphisms (SSCPs). Like restriction fragment length polymorphisms (RFLPs), SSCPs were found to be allelic variants of true Mendelian traits, and therefore they should be useful genetic markers. Moreover, SSCP analysis has the advantage over RFLP analysis that it can detect DNA polymorphisms and point mutations at a variety of positions in DNA fragments. Since DNA polymorphisms have been estimated to occur every few hundred nucleotides in the human genome, SSCPs may provide many genetic markers.

The nucleotide sequence of the melB gene coding for the melibiose carrier in Escherichia coli has been determined. The melibiose carrier is predicted to consist of 469 amino acid residues, resulting in a protein with a molecular weight of 52,029. The predicted carrier protein is highly hydrophobic (70% nonpolar amino acid residues). The hydropathic profile suggests that there are 10 long hydrophobic segments in the primary structure of the carrier protein. Most of them seem to traverse the membrane. Although the hydropathic profile of the melibiose carrier is similar to that of the lactose carrier as a whole, homology in the primary structure between the two carriers is very low. Furthermore, no homology in the nucleotide sequence is found in the structural genes for the two carriers. However, the nucleotide sequences of the intergenic regions are very similar between the melibiose operon and the lactose operon. There is a typical intercistronic regulatory sequence in the 3'-flanking region of the melB as well as in that of the lacY, which suggests the presence of another gene downstream of the melB.

The importance of negatively charged residues in transmembrane helices of many cation-coupled transporters has been widely demonstrated. Four Asp residues were located in the putative transmembrane helices of the Escherichia coli Na+/H+ antiporter, NhaA. We replaced each of these Asp residues by Asn in plasmid encoded nhaA and expressed these constructs in an E. coli mutant defective in both nhA and nhaB. Substitution of Asp-65 or Asp-282 (in the extramembrane region) had no effect on supporting the host mutant growth in the high NaCl- or LiCl-containing medium, and these two mutants had normal Na+/H+ and Li+/H+ antiporter activities. In contrast, substitution of Asp-133, Asp-163 or Asp-164 was detrimental to survival of the host mutant and impaired both Na+/H+ and Li+/H+ antiporter activities. These three Asp residues, conserved in the nhaA homologs from different species and which are located closely in the 3rd and 4th putative transmembrane helices, appear to play important roles in cation binding and transport.