Transcriptional activation of flanking sequences by Tn 1000 insertion

Tsuey Pin Lin, Erh Min Lai, Kin Yin To, Yu Sun Chang, Shih Tung Liu*

*Corresponding author for this work

Research output: Contribution to journalJournal Article peer-review

6 Scopus citations

Abstract

The carotenoid biosynthesis operon of Erwinia herbicola Eho13 consists of five genes, which are organized in the order crtE-crtX-crtY-crtI-crtB. These genes, with the exception of crtX, encode functions of β-carotene biosynthesis and give an orange-coloured phenotype in Escherichia coli. Since crtX is not involved in the biosynthesis of β-carotene, deletion of this gene does not alter the phenotype of pigmented cells. On the other hand, insertion of Tn 1000 into crtX or into the upstream untranslated region of the operon resulted in a light-yellow, rather than an unpigmented phenotype, indicating that Tn 1000 does not exert a strong polar effect when inserted in this operon. RNA analysis revealed that the sequence downstream from the insertion site was transcribed at a low level. Primer extension showed that the "-35"-like sequence in the terminal inverted repeats was not responsible for the transcription of the downstream sequence. Furthermore, primer extension and polymerase chain reaction (PCR) studies revealed that RNA transcribed from the promoters inside of Tn 1000 was extended through the terminal inverted repeats into the adjacent sequences. In addition Tn 1000, in either orientation, was able to generate fusion transcripts when placed upstream of a promoterless tetracycline-resistance gene and resulted in cells resistant to the drug. These results showed that Tn 1000 insertion transcriptionally activates the DNA sequences adjacent to the transposon.

Original languageEnglish
Pages (from-to)417-423
Number of pages7
JournalMGG Molecular & General Genetics
Volume245
Issue number4
DOIs
StatePublished - 07 1994

Keywords

  • Carotenoid biosynthesis genes
  • Erwinia herbicola
  • Tn 1000
  • Transcriptional activation

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