Determination of tubulin gene transcription start site in Stilonychia lemnae

Abstract

Contemporary literature concerning the regulation of transcription contains many discrepancies concerning o the precise position of the start site of transcription in the tubulin genes of hypotrychious ciliates. The reason for these inconsistencies lies in the methods applied by scientists. In the study presented, we have analyzed basic methods for the identification of the transcription start site for tubilin genes in a hypotrychious ciliate, Stylonychia lemnae. The modified method for the mapping of the transcription start site has been elaborated and is based on the analysis of sequencing and primer extension reaction products on a single gel. The use of nonphosphorylated primers in the sequence reaction (in accordance with the standard procedure) results in DNA fragments that have lower electrophoretic mobility than DNA fragments of the same size produced by primer extension. It complicates the interpretation of results and leads to possible mistakes. The differences in electrophoretic mobility may be avoided if primers phosphorylated by nonradioactive phosphorus are applied. In the present study, we used this method to map transcription start sites in α1-, α2-, β1- and β2-tubulin genes of S. lemnae.