Acanthamoeba castellanii

Strains, materials and methods

Strain description

Strain Neff (ATCC 30010).

Cell growth and media

Grown as a suspension culture at 30ºC in ATTC medium #354 (proteose peptone, glucose, vitamins, salts).

Purification of mtDNA

Isolation of mitochondria, CsCl/Hoechst dye gradient (by K.M. Lonergan in MWG's lab).

Cloning and sequencing

Cloning: tRNA cluster 1-rnl-tRNA cluster 2- rns-cox1/2: K.M. Lonergan (MWG's lab).
Entire genome: random fragments, generated by nebulization and fractionated according to size (0.5-1.0 kbp; 1.0-3.0 kbp), cloned in Bluescript KS+.

Sequencing strategy: Sanger sequencing of single-stranded DNA, using 35S label. Custom primers (13) used for limited primer walking. Mainly manual assembly, limited assembly using Staden's xbap.

Performed by: Sequencing of tRNA cluster 1-rnl-tRNA cluster 2-rns-cox1/2: K.M. Lonergan (MWG's lab). Cloning of complete genome: I. Plante (Unit). Sequencing of complete genome: I. Plante (Unit); rnl-rns region partially resequenced.

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