Localization of the in vivo and in vitro transcription initiation site and comparative analysis of the flanking sequences in the two main size classes of Ascaris lumbricoides rDNA.

摘要

An accurate in vitro transcription system which utilizes the cloned 8.8 and 8.4 kb size classes of Ascaris rRNA genes (pAlr8 and pAlr13) and two kinds of cellular extracts from Ascaris oogonia has been established. Both rDNA containing plasmids are efficiently transcribed in vitro by RNA polymerase I from a unique site of rDNA which corresponds to the in vivo initiation site. The in vitro transcription product has a triphosphorylated 5'-end and starts on a G localized 414 bp (pAlr8) upstream of the beginning of the mature 18S rRNA. The promoter region has been delimited by testing the in vitro template activity of a series of restriction fragments. The region essential for the accuracy of initiation is contained within nucleotides -72 to +65, but full efficiency of transcription requires the additional presence of the region from nucleotides +66 to +84. The sequences upstream from position -72 do not appear to modulate the efficiency of specific in vitro initiation. Furthermore, the sequences flanking the transcription initiation site from position -1500 to +570 have been determined in the two cloned representatives of the two rDNA main size classes.