Related Experiment Videos
Functional reverse transcriptases encoded by A-type mouse LINE-1: defining the minimal domain by deletion analysis
S L Martin1, J Li, L E Epperson
1University of Colorado School of Medicine, Department of Cellular, Structural Biology, Program in Molecular Biology, 4200 E. Ninth Avenue, Box B111, Denver, CO 80262, USA. martins@essex.uchsc.edu
Gene
|July 17, 1998
Summary
Mouse A-type LINE-1 elements possess reverse transcriptase activity, even with a previously suspected inactivating mutation. This study demonstrates functional capability of these retrotransposons, revealing that about half of the ORF2 sequence is needed for activity.
Area of Science:
- Molecular Biology
- Genetics
- Evolutionary Biology
Background:
- Long interspersed elements (LINEs) are retrotransposons that mobilize via RNA intermediates.
- The A-type LINE-1 (L1) subfamily in Mus species is a recently amplified variant.
- LINE-1 retrotransposition depends on the reverse transcriptase encoded by open reading frame 2 (ORF2).
Purpose of the Study:
- To assess the reverse transcriptase activity of ORF2 from three distinct mouse A-type L1 elements.
- To investigate the functional impact of a specific mutation thought to inactivate L1 reverse transcriptase.
- To determine the minimal functional domain of ORF2 required for reverse transcriptase activity.
Main Methods:
- Utilized a yeast genetic assay to measure reverse transcriptase activity.
- Tested ORF2 from a cDNA, a variant with a suspected inactivating mutation, and a hypothetical ancestral construct.
- Performed deletion analyses on ORF2 to map the minimal reverse transcriptase domain.
Main Results:
- All three tested A-type L1 elements exhibited reverse transcriptase activity in the yeast assay.
- A mutation previously believed to be inactivating did not abolish reverse transcriptase function.
- The L1 endonuclease domain is not essential for reverse transcriptase activity, and approximately 50% of ORF2 is sufficient for function.
Conclusions:
- Mouse A-type L1 elements encode functional reverse transcriptase.
- The identified mutation does not inactivate the enzyme's activity.
- A significant portion of the ORF2 coding sequence, excluding the endonuclease domain, is necessary for functional reverse transcriptase activity.