Strains, materials and methods
Species: Naegleria gruberi, C. Fulton strain NEG-M.
Source: ATCC # 30224
Cell growth and media
Cultured on solid freshwater ameoba medium, including malt and
yeast extract (ATCC medium # 997), at 25ºC. Performed by Tom Nerad at ATTC.
Purification of mtDNA
From 0.7 g wet wheight cells, total cellular DNA was extracted.
An A+T-rich DNA fraction
(mtDNA) was separated from nuclear DNA
(nDNA) by equilibrium centrifugation in CsCl-bisbenzimide gradients.
Random fragments, generated by nebulization
and size fractionation (0.5-1.0 kbp; 1.0-3.0 kbp) were
cloned into pFBS, a derivative of Bluescript KS+.
Performed by D. St-Louis (Unit).
Manual DNA sequencing was performed by the dideoxy chain termination method (Sanger et al. 1977), using
single-stranded DNA as templates and 35S-dATP as a label. Dried acrylamide gels were
autoradiographed and sequences were entered manually. One third of the readings were obtained with a LiCor automatic sequencer, including cycle sequencing
(Thermo Sequenase/7-deaza-dGTP, Amersham RPN 2438), using single-stranded
and plasmid DNA and a dye-labeled sequencing primer (IRD41).
Performed by Y. Zhu, I. Roewer, and D. St-Louis (Unit).
mtDNAs (FMGP)* | Other
complete mtDNAs | Gene
names | Mitochondrial Genomics ]