Transcription of a legumin gene from pea (Pisum sativum L.) in vitro.
The transcriptional activity of the pea legumin gene leg A in the HeLa cell lysate in-vitro transcription system has been assayed. Labelled transcripts were examined by hybridisation to restriction fragments of Leg A, and by glyoxalation followed by agarose gel electrophoresis. Although the legumin gene was not transcribed efficiently, transcripts were shown to be produced from the correct region of Leg A, and analysis by S1 nuclease mapping was used to show that the transcription start was the same as in vivo. The transcripts produced in vitro did not extent over the whole coding sequence of the gene; termination of transcripts occurred in a semi-random fashion. Transcription of templates truncated at their 3' ends (i.e. in coding sequence) showed that discrete transcripts were produced from the start to restriction sites at approx. +520 and +900 bases. Transcription of templates truncated at their 5' ends showed that sequences upstream of-97 bases relative to the transcription start had no appreciable effect on transcription in vitro.
Department of Botany, University of Durham, South Road, DH1 3LE, Durham, UK.
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Evans
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Bown
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Lycett
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Croy
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Boulter
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Gatehouse