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. 1997 Jul 8;94(14):7469–7474. doi: 10.1073/pnas.94.14.7469

Figure 4.

Figure 4

(A) RT and subsequent PCR amplification of the (modified) nitrate reductase mRNA. RNA was extracted from 20 Volvox spheroids of parent strain 153–48, wild-type algae (WT), or transformants HRN-1 through HRN-6 (RT-PCR of clone HRN-7 is not shown). As expected, RT-PCR with parent strain 153–48 yielded no correct product (19). Expression of nitrate reductase (NitA) is indicated by a plus or a minus. Sizes of PCR products (438 bp) were determined by using a 123-bp ladder as a size marker and by DNA sequencing. (B) Sequence of cDNA (438 bp), corresponding to nucleotides 1400–1837 of nitA sequence (18), obtained from RT and subsequent PCR amplification of the modified nitrate reductase gene harbored in Volvox clones HRN-1 and HRN-2, showing a homologous recombination event. Clones HRN-1 and HRN-2 resulted from transformation with plasmid 4. The sequence harbored in plasmid 4 is boxed (dashed line). The artificial base exchanges, with regard to the parent strain, are highlighted. Both introns (solid arrowheads) within this part of the cDNA were spliced correctly; in parent strain 153–48, splicing of the second intron is affected by mutation (G → A transition). The PCR primers used are indicated by horizontal arrows.