Functional reverse transcriptases encoded by A-type mouse LINE-1: defining the minimal domain by deletion analysis
| Autor: | B. Lieberman, L. E. Epperson, Sandra L. Martin, Jinfang Li |
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| Rok vydání: | 1998 |
| Předmět: |
Retroelements
Recombinant Fusion Proteins Retrotransposon Saccharomyces cerevisiae Biology Protein Engineering Transfection Fungal Proteins Mice Complementary DNA Genetics Animals Coding region Repetitive Sequences Nucleic Acid Sequence Deletion Binding Sites RNA-Directed DNA Polymerase Retroposon RNA General Medicine Molecular biology Reverse transcriptase Protein Structure Tertiary Long interspersed nuclear element |
| Zdroj: | Gene. 215:69-75 |
| ISSN: | 0378-1119 |
| DOI: | 10.1016/s0378-1119(98)00252-2 |
| Popis: | Long interspersed elements, or LINEs, are retrotransposons that move via an RNA intermediate. In mice, one polymorphic variant of L1 has amplified relatively recently, giving rise to the A-type subfamily in species belonging to the genus and subgenus Mus. Retrotransposition of LINE-1 (L1) requires the function of the L1-encoded reverse transcriptase that is produced from open reading frame 2 (ORF2). Here, we employ a convenient yeast genetic assay to determine the reverse transcriptase activity of the ORF2 obtained from three A-type L1 elements: one, a cDNA from the RNA in ribonucleoprotein particles; another with a purported inactivating mutation; and the third, a hypothetical ancestral construct. Because there are no examples of A-type elements that have transposed recently to inactivate a gene, this assay is the first step towards demonstrating the functional capability of mouse A-type LINE-1 elements. One of the three elements was believed to have been inactivated during evolution by the substitution of leucine for a highly conserved phenylalanine or tryptophan residue among known reverse transcriptases. This mutation did not inactivate the L1 reverse transcriptase in the yeast assay; thus, all three of the elements tested encoded reverse transcriptase activity. We further examined the minimal reverse transcriptase domain within ORF2 by creating a series of deletions. The results demonstrate that removal of the L1 endonuclease domain from the N-terminal region of ORF2 does not affect reverse transcriptase activity as determined by this assay, and that approximately half of the ORF2 coding sequence from mouse A-type L1 elements is required for functional reverse transcriptase. |
| Databáze: | OpenAIRE |
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