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Open Access Research article

Role of apoptosis-inducing factor (AIF) in programmed nuclear death during conjugation in Tetrahymena thermophila

Takahiko Akematsu* and Hiroshi Endoh

Author Affiliations

Division of Life Science, Graduate School of Natural Science and Technology, Kanazawa University, Shizenken, Kakuma-machi, Kanazawa, Ishikawa, Japan

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BMC Cell Biology 2010, 11:13  doi:10.1186/1471-2121-11-13

Published: 11 February 2010

Additional files

Additional file 1:

Mitochondrial nuclease activity. A. Purified mitochondria (2 μg protein) from ΔTTHERM_01104910 (a: lane 1) and ΔTTHERM_006222710 (b: lane 2) were incubated with 2 μg substrate plasmid DNA for 30 min at 37°C in 30 μl reaction buffer containing 20 mM KCl and 50 mM MOPS (pH 6.5). Lane 4 and 5 indicate 100-bp ladder size marker and λHindIII-digest, respectively. The substrate DNA appears in the nicked open circular (OC), linear (L), and supercoiled (SC) forms. B. The nuclease assay was performed under various incubation times. Lane 2-4 (a), substrate DNA was coincubated with ΔTTHERM_01104910 mitochondria. Lane 5-7 (b), substrate DNA was coincubated with ΔTTHERM_006222710 mitochondria. Lane 1 shows undigested sample.

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Additional file 2:

Cloning of TTHERM_01104910 and construction of the KO plasmid. A. One of AIF homologs (TTHERM_01104910), including the 1-kb 5' - and 3'-untranslated regions (UTRs), was amplified from CU428 genomic DNA using the following primers: A4910-F (5'-TTACCCTTCACTCAAGCC-3') and A4910-R (5'-ATGGTTGTGCTCGTAGTG-3'). Polymerase chain reaction (PCR) was carried out using the following program: 5 min at 94°C followed by 30 cycles of 94°C for 1 min, 53.5°C for 1 min, and 72°C for 5 min. The resulting 5,281-bp product was cloned into pT7 blue T-vector (Novagen) as a backbone for construction of the knock-out (KO) plasmid. Inverse PCR was performed using the backbone plasmid as template with the following primers: A4910-F-NotI (5'-gcggccgcGATCGACTCCAAGAGTCGAA-3') and A4910-R-XhoI (5'-ctcgagCTACTTACTTTGCCGC-3'). The start codon of this gene was destroyed by changing TAC to GAC in the forward primer. The PCR program included 5 min at 94°C followed by 30 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 8 min. The resulting 7,217-bp product was self-ligated and cloned. The plasmid was then digested with NotI and XhoI and integrated into the β-tubulin promoter (NotI/EcoRI-digested fragment) and Neor (EcoRI/XhoI-digested fragment) sites to express Neor under control of the β-tubulin promoter (Neor-cassette). B. The resultant plasmid (pKoTtA4910) was linearized with BamHI before biolistic bombardment.

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