Journal: Nucleic Acids Research
Article Title: Template-assisted synthesis of adenine-mutagenized cDNA by a retroelement protein complex
Figure Lengend Snippet: In vitro template-primed cDNA synthesis. ( A ) Bordetella bacteriophage DGR diversification of Mtd. mtd contains a variable region ( VR ), which encodes the receptor-binding site of the Mtd protein. Downstream of VR is the template region ( TR ). Adenines in TR (‘A’) are frequently replaced by another base in VR (‘N’). TR is transcribed to produce TR- RNA, which is then reverse transcribed to TR- cDNA. During this process, adenines in TR are mutagenized, as depicted by ‘X’ in TR -cDNA. Adenine-mutagenized TR- cDNA homes to and replaces VR , resulting in diversification of Mtd. bRT is the DGR reverse transcriptase, and avd the DGR accessory variability determinant. ( B ) Sequence elements of the 580 nt DGR RNA template used for reverse transcription reactions. ( C ) bRT-Avd, bRT, or Avd was incubated with the 580 nt DGR RNA and dNTPs, including [α- 32 P]dCTP, for 2h. Products resulting from the incubation were untreated (U), or treated with RNase (+R), DNase (+D), or both RNase and DNase (+R+D), and resolved by 8% denaturing polyacrylamide gel electrophoresis (PAGE). Lane T corresponds to internally-labeled 580 nt DGR RNA as a marker for the size of the template. The positions of the 580 nt band, and 120 and 90 nt cDNA bands are indicated. Nuclease-treated samples were loaded at twice the amount as untreated samples, here and throughout unless otherwise indicated. Lane M here and throughout corresponds to radiolabeled, single-stranded DNA molecular mass markers (nt units). ( D ) DGR RNA templates containing internal truncations in TR . ( E ) Radiolabeled cDNA products resulting from bRT-Avd activity for 2 h with intact (WT) or internally truncated 580 nt DGR RNA as template. Samples were treated with RNase and resolved by denaturing PAGE. The positions of the 120 and 90 nt cDNAs produced from intact template are indicated by red and yellow circles, respectively, as are positions of the correspondingly shorter cDNAs produced from truncated RNA templates. ( F ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template. Prior to reverse transcription, the RNA template was mock-treated (–Per) or treated with periodate (+Per). Products of the reaction were untreated (U) or treated with RNase (+R), and resolved by 4% (top) or 8% (bottom) denaturing PAGE. In the top gel, the red arrowhead indicates the ∼580 nt species, and the green arrowheads the several ∼540 nt species. In the bottom gel, the black arrowheads indicate the 120 and 90 nt cDNA products. The black vertical line within the gel indicates irrelevant lanes that were removed for display purposes. A 2-fold higher quantity was loaded for +Per samples than –Per samples.
Article Snippet: Reverse transcription reactions with HIV-1 RT were carried out as above, except in 10 μl and containing 10 units RNase inhibitor (NEB), 0.1 μCi/μl [α-32 P]dCTP, 30 ng/μl RNA template, 1 μM PG117 primer ( ) and 2 units of HIV-1 RT (Worthington Biochemical), and the reaction was carried out for 30 min.
Techniques: In Vitro, Binding Assay, Sequencing, Incubation, Polyacrylamide Gel Electrophoresis, Labeling, Marker, Activity Assay, Produced