Porphyra purpurea

Strains, materials and methods

Culture and media

Porphyra purpurea gametophytes were cultured in modified D-11 seawater medium at 15ºC, under a 16 h:8 h LD cycle (Mitman and van der Meer, 1994).

Purification of mtDNA

Total cellular DNA was extracted from P. purpurea gametophytes by the method of Doyle and Doyle (1990). An A+T-rich DNA fraction (consisting of a mixture of mitochondrial and chloroplast DNAs) was separated from nuclear DNA by equilibrium centrifugation in CsCl-bisbenzimide gradients (Douglas, 1988).

Cloning

The mtDNA was isolated by agarose gel electrophoresis after cutting the A+T-rich fraction with SacI. The largest (35 kbp) restriction fragment was the linearized mtDNA (which has only 1 SacI site), whereas the ptDNA was cut into several fragments of <15 kbp (M. Reith and J. Munholland, 1993). Electroeluted mtDNA was physically fragmented by nebulization, size-selected (500-3000 bp), end-polished and cloned into the SmaI site of pBluescript II KS+ (Stratagene). Recombinant plasmids containg mtDNA inserts were identified by colony hybridization using the 35-kbp SacI restriction fragment (see above) as a probe.
Performed by I. Plante and D. St-Louis (Unit).

Sequencing

DNA sequencing was performed by the dideoxy chain termination method (Sanger et al., 1977), using single-stranded DNA as template and ³⁵S-dATP as a label. Dried acrylamide gels were autoradiographed and sequences were entered manually.
Performed by D. St-Louis (Unit).