Detecting DNA cytosine methylation using nanopore sequencing.

In nanopore sequencing devices, electrolytic current signals are sensitive to base modifications, such as 5-methylcytosine (5-mC). Here we quantified the strength of this effect for the Oxford Nanopore Technologies MinION sequencer. By using synthetically methylated DNA, we were able to train a hidden Markov model to distinguish 5-mC from unmethylated cytosine. We applied our method to sequence the methylome of human DNA, without requiring special steps for library preparation.

Tumor cell splice variants of the transcription factor TEF-1 induced by SV40 T-antigen transformation.

The large tumor antigen (TAg) of simian virus 40 is able to transform cells through interactions with cellular proteins, notably p53 and Rb. Among the other proteins that form complexes with TAg is TEF-1, a transcription factor utilized by the viral enhancer to activate expression of the early gene which encodes TAg. We show that fibroblasts contain several alternately spliced TEF-1 mRNAs, the most abundant of which encodes a protein with an additional four amino acid exon compared to the database entry for Hela cell TEF-1. Transformation by TAg induces alternate splicing, producing a more abundant form lacking this exon and matching the published sequence. Splicing variants lacking this exon were detected in mouse pancreatic tumors and in cell lines derived from human pancreatic cancers, in contrast to a single isoform with the exon in normal mouse pancreas. A total of eight splice variants were identified, with the loss of the four amino acid exon typical of transformed cells. These and other data presented suggest that TAg 're-models' host cell transcription factors that are used early in viral infection, and thereby mimics an event that naturally occurs during transformation. The data indicate that TEF-1 alterations may be a hallmark feature of tumorigenesis.