FIG. 3.

Both Sec11a and Sec11b are expressed in H. volcanii, but only Sec11b is essential. A. RNA was extracted from H. volcanii cells grown to exponential phase and used to direct the synthesis of cDNA. PCR was then performed in the absence of nucleic acids (−) (lanes 1) or in the presence of genomic DNA (+DNA) (lanes 2), total RNA (+RNA) (lanes 3), or cDNA (+cDNA) (lanes 4), using primers against either sec11a (left panel) or sec11b (right panel). B. Schematic diagram of the plasmids used to delete the Sec11a- and Sec11b-encoding genes. C. PCR amplification of H. volcanii cells transformed with plasmid pIDT-Sec11a or pIDT-Sec11b, designed to delete either sec11a or sec11b, respectively. The arrows show the positions of the PCR amplification product encoding the Sec11a or Sec11b flanking regions separated by the H. volcanii trpA gene, presented in the last lane of each panel. Also shown in each panel are PCR products obtained using genomic DNA (lanes 1), genomic DNA including the integrated pIDT-Sec11a or pIDT-Sec11b plasmids (lanes 2), and the same DNA taken from cells grown in tryptophan-free, 5-FOA- and uracil-containing medium, i.e., following expulsion of the plasmid and native Sec11a- or Sec11b-encoding gene (lanes 3) as template. In the right panel, lane 4 shows the PCR product of the cells considered in lane 3 but returned to tryptophan-containing medium such that DNA encoding the sec11a or sec11b flanking regions separated by the H. volcanii trpA gene is expelled from the genome with the plasmid. D. RNA was extracted from H. volcanii cells from which the Sec11a-encoding gene had been deleted and used to direct the synthesis of cDNA. PCR was then performed in the absence of nucleic acids (−) (lane 1) or in the presence of genomic DNA taken from untreated cells (+DNA) (lane 2) or total RNA (+RNA) (lane 3) or cDNA (+cDNA) (lane 4) from the deletion strain, using primers against either sec11a (left panel) or sec11b (right panel).