Enhancer and Silencer Elements
Conserved Sequences in Eukaryotic Promoters
Trans-Acting Factors Control Gene Expression
Cloning A Plant Trans-Acting Factor
Regulatory Genes As Trans-Acting Factors
Regulatory Sequences Control Gene Expression
Not all genes are turned on at all times during the life cycle of a plant. Different genes are required for the completion of different steps in the developmental and sexual maturation of the plant. Two general types of control can be described.Temporal regulation - a type of regulation of gene expression in which a gene is only expressed at a specific time in development; for example, genes that are only expressed in the light or during flower development
Spatial regulation - a type of regulation of gene expression in which a gene is only expressed in a specific location in the plant; for example, seed storage proteins
Although we can distinguish between these two types of regulation, many genes will actually fall into both classes. For example, seed storage proteins are only expressed in the seed, but they also are only expressed during a short period of time during the development of the seed. Furthermore, because the binding of RNA Polymerase II to the promoter is the key step in gene expression, it follows that sequences may exist in the promoter that control temporal and spatial gene expression. Can those sequences by defined?
Specific Promoter Sequences Are Required For Regulated Gene Expression
The first example of the dissection of a plant promter to identify sequences responsible for gene expression was published in 1985 (Nature 315:200). This paper specifically reported on the effects of deletions of 1052 bases of the pea rbcS-E9 promoter. rbcS is the nuclear gene that encodes the small subunit of RUBISCO. The authors analyzed the promoter by creating deletions of the promter and analyzing the effects of the deletions in transgenic plants. The following table summarizes their results. It should be noted that the authors measured changes in rbcS gene expression for each truncated promoter by comparing its expression to the constitutively expressed NPTII reporter gene.
| Deletion | Effect on expression |
|---|---|
| -1052 to -437 | 3X reduction |
| -1052 to -352 | 3X reduction |
| -1052 to -35 | 6X reduction |
| -1052 to -14 | no expression |
| -107 to -56 | 2x increase (CAAT box not important) |
Thus, sequences between 0 and -35 control light responsiveness. But can the size of the relevant element be defined further. They next made the following construction and analyzed its effect on transgenic plants.
--1052 to -2 of promoter --- CAT --- NOS 3'
This construction give light mediated expression of the CAT mRNA. Thus the boundaries of the light responsive element could be set at -35 to -2.
Hormone Responsiveness Sequences Can Be Indentified
The plant hormone ethylene causes a dramatic change in the expression of certain genes in the plant. One gene that responds to ethylene is chitinase. This gene appears to be involved in the plant defense response, but more specifically it may be part of a repertoire of genes that are induced during stress. For example, treatment of bean plants with mercuric chloride induces high levels of chitinase expression. Growing bean plants in an ethylene environment will also induce the chitinase gene to high levels of expression. Therefore, it was thought that ethylene may be the mediator for the expression of chitinase and other stress related proteins. But this does not appear to be the case. But an important question is what sequences are required for ethylene responsiveness?The promoter sequences responsible for the ethylene response of bean chitinase were determined in 1989 (The Plant Cell 1:599). Bal31 deletions of the promoter were made and their effect on gene expression were measured in transgenic tobacco plants. A comparison of two bean chitinases genes showed that they were ethylene inducible and revealed two regions that were homologous in the two promoters. Region I is 96% homologous between the two genes, and a second region, Region II (-181 to -238) is completely homologous in 13 and 43 bp section. The following conclusions were drawn from the transgenic plants.
- deletion of region I had no effect on ethylene induced gene expression
- removal of sequences between -1057 and -846 resulted in a three fold increase in expression
- removal of sequences between -846 and -422 resulted in a twenty fold decrease in expression, but ethylene expression is still detected
- removal of sequences between -422 and -195 eliminated ethylene responsiveness; interestingly, this deletions falls in the middle of region II
These experiments prove that sequences required for hormone induction can be found in promoters of genes.
Copyright © 1998. Phillip McClean