Journal: Eukaryotic Cell
Article Title: Relaxed Primer Specificity Associated with Reverse Transcriptases Encoded by the pFOXC Retroplasmids of Fusarium oxysporum
Figure Lengend Snippet: Exogenous reverse transcription reactions with MN-treated pFOXC RT and MMLV RT. Reactions with mtRNP particles isolated from pFOXC3-containing strains digested with MN (lanes 1 to 5) or MMLV RT (lanes 7 to 10). Lane 1, no exogenous RNA. Lanes 2 to 5 and 7 to 10, reaction mixtures containing 93-nt C3-2R RNA that corresponds to the 3′ terminus of the pFOXC3 plasmid transcript. Reactions were carried out with (lanes 4, 5, 9, and 10) or without (lanes 2, 3, 7, and 8) a 34-nt oligonucleotide that is complementary to a 25-nt region at the 3′ end of the RNA template. Following cDNA synthesis, products were incubated with RNase A (lanes 3, 5, 8, and 10) or left untreated (lanes 1, 2, 4, 7, and 9), prior to electrophoresis in a 6% polyacrylamide gel containing 8 M urea. Numbers on the left indicate the sizes (nucleotides) of a 100-bp marker and Sau3AI fragments of pBS(−) molecular weight markers (M, lane 6). Numbers on the right indicate the sizes (in nucleotides) of the 32 P-labeled DNA products as well as a schematic drawing of the most favorable base-pairing interactions of snapped-back C3-2R RNAs that may correspond to the products observed (dashed line, C3-2R RNA; solid line, cDNA; open box, 2R oligonucleotide). Potential base-pairing interactions of the 2R oligonucleotide self-dimer that is extended by the pFOXC RT are shown at the bottom, with the incorporated nucleotides indicated in lowercase letters.
Article Snippet: Interestingly, the major cDNA products obtained with MMLV RT were significantly shorter (73, 76, and 82 nt) than those obtained with the MN-treated pFOXC RT.
Techniques: Isolation, Plasmid Preparation, Incubation, Electrophoresis, Marker, Molecular Weight, Labeling