Reverse transcriptase activity from human embryonal carcinoma cells NTera2D1.
We have identified an RNA-dependent DNA polymerase activity in the microsomal fraction of human pluri-potential embryonal carcinoma cells NTera2D1, which are known to express the full length coding strand of the genomic Line-1 (L1) elements. This activity was classified as a reverse transcriptase (RT) based on its utilization of an RT specific synthetic poly(Cm) template in the presence of Mn2+ ions. Treatment of the cell by ultraviolet irradiation (200 erg/mm2) which resulted in a 2- to 3-fold enhancement of the RT activity, was required for the reproducible detection of the activity throughout the entire purification procedure. More than a 100-fold enrichment in RT activity was obtained by centrifugation in a glycerol step gradient and a linear sucrose density gradient followed by Sephacryl S-1000 gel filtration. These experiments demonstrated that the RT activity was associated with a macromolecular complex having the characteristics of a viral-like particle with a major protein component of 37 kd. The presence of L1 mRNA in RT-containing fractions suggests that the activity identified could originate from L1 elements and/or be involved in the mechanism of retroposition.
Service de Génétique Médicale, Hôpital Ste-Justine, Université de Montréal, Québec, Canada.
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Deragon
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Sinnett
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Labuda
D001483:Base Sequence; D002460:Cell Line; D006801:Humans; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000528:radiation effects; D013724:Teratoma / Q000201:enzymology; D014466:Ultraviolet Rays
D015345:Oligonucleotide Probes; D012194:RNA-Directed DNA Polymerase