Submission Type
Poster
Abstract
Have you ever wondered what causes diseases like hemophilia, muscular dystrophy, or even cancer? The culprit may be lying in your genome: Transposable Elements (TEs) are genetic elements that replicate and move positions within a genome. Almost half of the human genome is comprised of TEs, with 17% being a specific type of TE called Long Interspersed Nuclear Elements (LINEs), which insert via Target Primed Reverse Transcription (TPRT). Studying these elements will lead to a greater understanding of both genome dynamics and genetic delivery tools. This study uses the R2 LINE element from Bombyx mori (R2Bm) which has an Open Reading Frame (ORF) that encodes Zinc-Finger (ZF), Myb, Reverse Transcriptase (RT), and a DNA endonuclease domain. We hypothesize that domain 6a is involved in binding to host DNA and the thumb in binding to both DNA and RNA. To test this hypothesis, conserved residues within these regions were mutated to alanine. Wild type and mutant R2Bm proteins were expressed in BL21 cells and purified using affinity chromatography. Purified proteins were run on SDS-PAGE gels and the amounts were quantified using a Bovine Serum Albumin (BSA) standard curve. We were able to successfully purify proteins from the domain 6a mutant and the wild type. For the nucleic acid binding experiments, 5' and 3' PBM RNA were successfully synthesized using in vitro transcription. While experiments are ongoing, we have determined that the K675 and K676 residues from domain 6a are involved in DNA binding.
Recommended Citation
Cawley, Michaela; Farner, Jordyn; and Geraghty, Meghan, "207 - Mutations in the Reverse Transcriptase and Thumb of R2Bm Proteins and Their Impact on Nucleic Acid Binding" (2025). GREAT Day Posters. 60.
https://knightscholar.geneseo.edu/great-day-symposium/great-day-2025/posters-2025/60
207 - Mutations in the Reverse Transcriptase and Thumb of R2Bm Proteins and Their Impact on Nucleic Acid Binding
Have you ever wondered what causes diseases like hemophilia, muscular dystrophy, or even cancer? The culprit may be lying in your genome: Transposable Elements (TEs) are genetic elements that replicate and move positions within a genome. Almost half of the human genome is comprised of TEs, with 17% being a specific type of TE called Long Interspersed Nuclear Elements (LINEs), which insert via Target Primed Reverse Transcription (TPRT). Studying these elements will lead to a greater understanding of both genome dynamics and genetic delivery tools. This study uses the R2 LINE element from Bombyx mori (R2Bm) which has an Open Reading Frame (ORF) that encodes Zinc-Finger (ZF), Myb, Reverse Transcriptase (RT), and a DNA endonuclease domain. We hypothesize that domain 6a is involved in binding to host DNA and the thumb in binding to both DNA and RNA. To test this hypothesis, conserved residues within these regions were mutated to alanine. Wild type and mutant R2Bm proteins were expressed in BL21 cells and purified using affinity chromatography. Purified proteins were run on SDS-PAGE gels and the amounts were quantified using a Bovine Serum Albumin (BSA) standard curve. We were able to successfully purify proteins from the domain 6a mutant and the wild type. For the nucleic acid binding experiments, 5' and 3' PBM RNA were successfully synthesized using in vitro transcription. While experiments are ongoing, we have determined that the K675 and K676 residues from domain 6a are involved in DNA binding.
Comments
Sponsored by Varuni Jamburuthugoda