Flow chart, linearized map of mtDNA control region, and single CD34⁺ clones. (A) Mononuclear cells from BM and CB were separated by density gradient centrifugation and washed twice in phosphate-buffered saline. CD34⁺ cells were sorted by single cell deposition into 96-well microtiter plates using a phycoerythrin anti-CD34 monoclonal antibody (BD Biosciences), a MoFlo cytometer, and a CyClone automated cloner (Dako-Cytomation) in the 0.5 single drop mode. After 5 days of culture in media containing stem cell factor, Flt-3, thrombopoietin, and G-CSF, each well of the microplate was carefully examined and scored for cell number. To directly sequence the control region of mtDNA, DNA in these colonies, derived from single CD34⁺ cells, was subjected to nested gene amplification (see “Materials and methods”). Sequencing was performed on an ABI Prism 3100 Genetic Analyzer in both orientations. Evidence of mtDNA heterogeneity was further confirmed by reamplification of the original lysate. (B) Linearized map and function location of mtDNA control region between nucleotides 16 024 to 16 569 and 1 to 576 (D-loop); HV1 (hypervariable segment 1, nucleotides 16 024 to 16 383), HV2 (hypervariable segment 2, nucleotides 57 to 372), OH (H-stand origin, nucleotides 110 to 441), CSB (conserved sequence block, nucleotides 213 to 235, nucleotides 299 to 315, nucleotides 346 to 363), mt5 (control element, nucleotides 16 194 to 16 208), mt3L (L-strand control element, nucleotides 16 499 to 16 506), TAS (termination-associated sequence, nucleotides 16 157 to 16 172), PL (L-strand promoter, nucleotides 392 to 345), PH1 (major H-strand promoter, nucleotides 545 to 567), TFB (mitochondrial transcription factor binding site, nucleotides 233 to 260, 276 to 303, 418 to 445, 523 to 550), mt4H (H-strand control element, nucleotides 371 to 379), mt3H (H-strand control element, nucleotides 384 to 391), and 7S DNA (nucleotides 16 106 to 16 191). The homopolymeric C tracts located on HV1 (nucleotides 16 184 to 16 193; 5CT4C) and HV2 (nucleotides 303 to 315; 7CT5C). (C) The number and morphology of CD34⁺ clones after 5-day suspension culture. Each picture (original magnification, × 200) shows a different grade: (i) 5 or fewer cells per well, grade 1; (ii) 6 to 10 cells per well, grade 2; (iii) 11 to 20 cells per well, grade 3; and (iv) 21 or more cells per well, grade 4.