Plasmid vectors designed for the analysis of transcription termination signals

Alik Honigman*, Jamal Mahajna, Shoshy Altuvia, Simi Koby, Dina Teff, Hilla Locker-Giladi, Hana Hyman, Chanoch Kronman, Amos B. Oppenheim

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

8 Scopus citations

Abstract

We have constructed synthetic operons in which two genes (cat and lacZ or cat and galK) were placed in tandem under the control of the bacteriophage λ oLpL operator and promoter. Restriction sites were introduced between the promoter and the proximal cat gene or between the cat and lacZ or galK genes. In the latter case, introduction of a transcriptional terminator between the two structural genes should affect only the distal gene. Thus, following induction, the expression of the cat gene serves as an internal control, compensating for changes due to plasmid copy number or possible decrease in transcription initiation. We used these plasmids to select a λ DNA fragment which includes the N-unresponsive tJ transcriptional terminator. This DNA fragment was inserted between the cat and galK genes. Enzymatic assays of these two gene activities following induction indicate that transcripts initiated at the pL promoter under N+ conditions terminate at tJ between the two genes. S1-nuclease analysis showed that these transcripts terminate at several sites in the tJ region. Similar results were obtained whether the host cells were RNaseIII+ or RNaseIII-. As a control, we showed a complete antitermination of the λ t'I terminator under similar conditions, indicating that a sufficient amount of the N gene product is made from one N gene copy to suppress terminators carried on multicopy plasmids.

Original languageAmerican English
Pages (from-to)131-141
Number of pages11
JournalGene
Volume36
Issue number1-2
DOIs
StatePublished - 1985

Bibliographical note

Funding Information:
We thank Drs. Mike Berman,D on Cow, Max GottesmanJ,i m Lautenbergearn dAvigdor ShafTer-man for bacterials trainsa nd plasmids.W e thank Dr. Don Court for criticalr eviewo f the rn~usc~pt andA lisa Shilor and DarleneW hitef or theirh elpi n bringingt hism anuscriptto its final form. This work was supportedin part by the Fund for Basic Research administeredb y the Israel Academy of Sciencesa nd Humanities.

Keywords

  • Gene regulation
  • N-unresponsive t terminator
  • hybrid operons
  • promoter
  • recombinant DNA

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