title: Terminal telomere repeats are actually short in telomerase-negative immortal human cells.
abstract: Telomerase-negative immortal human cells maintained telomere length by a mechanism called alternative lengthening of telomeres (ALT mechanism). These cells (ALT cells) have two prominent characteristics of long telomere repeats at each chromosome end revealed by Southern blotting (terminal restriction fragments: TRF) and the presence extrachromosomal telomere repeat (ECTR) DNA. We report here that the TRF length of ALT cells revealed by the conventional unidirectional (UD) current or pulse-field (PF) current electrophoresis appeared to be over estimated. The TRF length determined by the pulse inverse-field (PIF) current electrophoresis (2-9 kbp depending upon cell lines) was much smaller than that (ca. 23 kbp) by UD or PF current electrophoresis. These results were in consistent with very weak telomere staining in situ at chromosome ends in ALT cells. When a mixture of HinfI-digested genomic DNA of human diploid fibroblasts and synthetic telomere repeat DNA with similar size of ECTR DNA was electrophoresed using a UD current, the apparent TRF size shifted to larger molecular weight, while the size shift did not occur by PIF current electrophoresis. These results together with other data indicate that the unusually long TRF of ALT cells determined by using conventional electrophoresis is an artifact produced by a complex formed by short TRF and short ECTR DNA.
authors.affiliation: Department of Cellular and Molecular Biology, Graduate School of Biomedical Sciences, Hiroshima University, Japan.
authors.forename: Tohru
authors.initials: T
authors.lastname: Higaki
authors.forename: Taro
authors.initials: T
authors.lastname: Watanabe
authors.forename: Ichiro
authors.initials: I
authors.lastname: Tamatomi
authors.forename: Hidetoshi
authors.initials: H
authors.lastname: Tahara
authors.forename: Masanobu
authors.initials: M
authors.lastname: Sugimoto
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Furuichi
authors.forename: Toshinori
authors.initials: T
authors.lastname: Ide
mesh_terms: D002460:Cell Line; D002461:Cell Line, Transformed; D006801:Humans; D019098:Telomerase / Q000032:analysis / Q000235:genetics*; D016615:Telomere / Q000737:chemistry / Q000235:genetics*; D020079:Terminal Repeat Sequences / Q000235:genetics*
chemical_list: D019098:Telomerase