Regulatory Sequences Control Gene Expression

Enhancer and Silencer Elements

Role of 3' Sequences

Role of Introns

Conserved Sequences in Eukaryotic Promoters

Trans-Acting Factors Control Gene Expression

Cloning A Plant Trans-Acting Factor

Regulatory Genes As Trans-Acting Factors

Tissue-Specific Binding Of Trans-Acting Factors

Course Topics

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Role of Introns in Gene Expression

Before we discuss experiments that study the effect of introns on gene expression, we need to talk about transient assays, another tool used for gene expression studies. With this technique, a plasmid carrying a specific gene construction is introduced into plant protoplasts, and the expression of the genes on that plasmid are monitored within hours after the experiments begins. The genes are introduced into the cells by electroporation or PEG (polyethylene glycol) treatment which disrupts the integrity of the cell membrane. These treatments are performed in a media that contains the plasmid that will be monitored. After the treatment, pores form in the membrane for a period of time. While there are pores in the membrane, DNA can move into the cells. Once the membranes seals, the DNA is in the cell and machinery of the cell can express the genes on the plasmid. This technique allows the scientist to rapidly compare the effects of different DNA sequences on gene expression.

The effect of intron 1 from maize gene sucrose synthetase (shrunken) gene was was analyzed in 1989 (Plant Physiol. 91:1575). This intron was introduced between either the CaMV 35S promoter or the shrunken promoter and the CAT reporter gene. The intron was placed in the normal or reverse orientation. Control plasmids contained the promoters without the intron. Each of these plasmids were then introduced into protoplasts of several grass species, and the level of CAT expression was monitored with transient assays. The results are presented in the following table.

Intron, Orientation Promoter Species Expression level relative to control:
Shrunken, normal Shrunken Panicum 6-57 X increase
Shrunken, normal 35S Panicum 22-27 X increase
Shrunken, normal Shrunken Pennisetum 2 X increase
Shrunken, normal 35S Pennisetum 11 X increase
Shrunken, normal Shrunken Maize 1-172 X increase
Shrunken, normal 35S Maize 30-90 X increase

These experiments demonstrate that the intron can have a positive effect on gene expression. But this effect was only noticed when the intron was in the normal orientation. When the orientation was reversed, gene expression was not noticed. Also the increase was most dramatic when the intron was linked to the CaMV 35S promoter, and it was greatest in maize cells.

Copyright © 1998. Phillip McClean