Figure 1.
Expression of human β1,4-galactosyltransferase (hGT) gene in tobacco BY2 cells. (A) The recombinant hGT plasmid for expression of human β1,4-galactosyltransferase in tobacco cells. The hGT coding region lies downstream of the CaMV35S promoter, followed by the nopaline synthase terminator (nos-t) from pBI221. The construct also has the neomycin phosphotransferase II (nptII) gene under the control of the nopaline synthase promoter (nos-p) from pGA482. RB/LB, right and left borders, respectively. (B) Southern blot analyses of genomic DNAs from transformed and wild-type BY2 tobacco cells. DNAs were digested with HindIII and EcoRI and probed with a 32P-labeled hGT gene fragment as described in Materials and Methods. WT, wild type; 1, 4, 5, 6, 8, and 9, cell line number for transformed tobacco cells. The numbers to the left indicate the sizes (kbp) and positions of λ DNA fragments digested with HindIII. Size of the hybridizing fragment (2.2 kbp) is indicated. (C) Western blots of immunoreactive proteins from transgenic and nontransgenic tobacco cells. Proteins were denatured, resolved by SDS/PAGE, and electroblotted onto a nitrocellulose membrane. The blots were probed with anti-hGT antibody as described in Materials and Methods. Lanes contain proteins from cell extracts of cell lines 1, 6, 8, 9 and wild type and microsome fractions of cell lines 1, 6, 8, 9 and wild type. Molecular mass of marker proteins are indicated on the left.