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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The Effect Of Ennhancer And Silencer Elements On Gene Expression

Enhancers

Certain sequences have been described that effect the level of gene expression. We have already described some that have an up and down effect on expression. But other sequences have been found that have a dramatic effect on expression.

Enhancer- a DNA sequence that greatly increases the expression of a gene in its vicinity; the sequence can reside upstream or downstream and its orientation is not fixed; these sequences have been shown to have an effect as far away as 3000 bp

The effect of enhancers elements were studied in the following two papers: PNAS 83:8560 (1986) and EMBO Journal 7:297 (1988). Alpha-conglycinin is a seed storage protein of soybean that is constructed from several subunits. The genes for these subunits are expressed at high levels during specific stages of seed development. What effect do the different promoter sequences of these genes have on seed development. Again, as with other researchers, deletions of the promoter of the gene for one subunit (alpha') were constructed and analyzed in transgenic plants. The results were:

  • Deletions of the promoter to -208 did not eliminate expression, although about a two-fold reduction was seen with the last deletion.
  • Deletion through -159 nearly eliminates expression.
  • Further deletions into the gene eliminate all expression.
Although this experiment showed that expression was controlled by promoter sequences, it could not be determined whether this control was at the level of general expression or whether seed-specific expression was affected. Further experiments were performed. Since these experiments showed that sequences within 300 bp of the promoter were essential for expression, these sequences were used in a reporter system. The following control construct was made:

CaMV 35S Promoter --- CAT --- Alpha ' 3' end

The -78 to -257 region of the promoter was then inserted into different locations of this construct, and the effects on gene expression were monitored. The following results were obtained from transgenic tobacco plants.

  • Without the promoter insertion, CAT activity was detected in all tissues equally.
  • Insertion of the element into the promoter resulted in seed-specific enhancement of expression. This enhancement occurred regardless of the orientation.
  • Insertion of the element, again in either orientation, before the 3' end of the gene increased seed expression but not to the level seen when the insertion was made into the promoter.
  • Insertion of the element beyond the 3' element did not increase seed expression.
  • The element appears to act as an enhancer.

Silencers

The experiments that analyzed the promoter of the alpha' subunit of the soybean beta conglycinin protein revealed that sequences were present that exhibited enhancer-like activities as well as temporal and spatial regulation. Spatial regulation is of particular importance, since, for example, there is no need to express a seed storage protein in the leaf of a plant or a protein involved in photosynthesis in the root of a plant. How is this negative regulation manifested? Again, reporter genes driven by different promoters were analyzed in transgenic plants.

The effects of a promoter element from the pea chlorophyll a/b binding protein gene on gene expression was reported in 1986 (Nature 323:551). Previous experiments had shown that kanamycin resistance could be obtained only if the first 400 bp of the promoter were present driving an NPT II resistance gene. If sequences from -100 to -347 were deleted then kanamycin resistance could not be obtained which suggested this region contained a positive regulatory element involved in tissue-specific or light dependent expression. This region was then introduced into transgenic plants linked to a reporter gene. The following constructs were made and their effect on gene expression are noted in the following table.

Expression level in:
Construct Sequence Light Dark Root
pABN1 [-347 cab promoter -100]-NOS Promoter--NPTII--NOS 3' 4-8X 1X 0
pABN2 [-100 cab promoter -347]-NOS Promoter--NPT II--NOS 3' 5-8X 1X 0
pABN3 2X[-347 cab promoter -100]-NOS Promoter--NPT II--NOS 3' 10-13X 1X 0
NOS Control NOS Promoter--NPT II--NOS 3' 1X 1X 1X

The following conclusions can be made from the results presented in the table.

  • The -100 to -347 element contains sequences which act as an enhancer since gene expression is increased, and the orientation of the element does not matter (pABN1, pANB2).
  • The element expresses an additive effect since gene expression is greater when two copies of the element are present (pABN3).
  • The element has silencing activity in the root since no expression is detected in the root when it is present (pABN1, pABN2, pABN3).
  • The element controls light regulation since light is required for the enhanced levels of expression (pABN1, pABN2, pABN3).
Copyright © 1998. Phillip McClean