Cloning: A mtDNA-enriched fraction was either (i) partially digested with Sau3AI or EcoRI and then used for the construction of phage libraries in lambda EMBL3 and EMBL4 (Stratagene), respectively (Wolff et al., 1993) or (ii) cut with HindIII or SalI and cloned in pUC9 (Yanish-Perron et al., 1985) and pBluescriptII KS+ (Stratagene). Recombinant phages and plasmids carrying mitochondrial inserts were identified and mapped by using them as probes in Southern hybridization experiments against P. wickerhamii extranuclear DNA. Mapping and restriction analysis revealed that seven segments of 0.2 to 2.6 kbp length (about 5 kbp in total) were not covered by the clones in the four libraries. Therefore, seven pairs of primers were synthesized to amplify the uncloned regions by the polymerase chain reaction (PCR), using Replitherm polymerase (Biozym). The resulting PCR products were cloned into pBluescript and identified by hybridization.
Performed by: Cloning of large mtDNA fragments was done by Gabi Wolff in Bochum. All subclone libraries were constructed by I. Plante (Unit).
Sequencing strategy: For DNA sequencing by the dideoxy chain termination method (Sanger et al., 1977), random subclone libraries were generated by digesting the cloned mtDNA independently with Sau3AI, RsaI, TaqI, HinPI, HpaII or by physical fragmentation with a nebulizer (Surzycki, Indiana University, Bloomington, Indiana, USA), and cloning into pBluescript II KS+. Recombinant clones were identified by colony hybridisation and probing with a mtDNA-enriched fraction. Single-stranded DNA was obtained by superinfecting the clones with helper phage K07 (Vieira & Messing, 1987). High resolution polyacrylamide gel electrophoresis was performed for long-range reading (Lang BF, Burger G. 1990. Anal Biochem 188: 176-180). Both strands were sequenced, and in regions where clone coverage was insufficient, specific sequencing primers were synthesized for limited "primer walking".
Performed by: The complete sequence was determined in the OGMP Sequencing Unit by I. Plante and G. Wolff (visitor in the Unit for 6 months). The mitochondrial rns and rnl and 75% of cox1 were sequenced independently in Bochum by G. Wolff.