Method of increasing transcription with nucleic acids...

Details Method of increasing transcription with nucleic acids... Method of increasing transcription with nucleic acids...

2001-04-19

2002-10-29

McKelvey, Terry (Department: 1636)

Chemistry: molecular biology and microbiology

Process of mutation, cell fusion, or genetic modification

Introduction of a polynucleotide molecule into or...

C435S468000, C435S471000, C435S375000

Reexamination Certificate

active

06472211

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates generally to gene expression and specifically to a novel enhancer element that increases the rate of transcription of a gene operably linked thereto, particularly in plants.
BACKGROUND OF THE INVENTION
Genes are regulated in an inducible, cell type-specific or constitutive manner. There are different types of structural elements which are involved in the regulation of gene expression. Cis-acting elements, located in the proximity of, or within genes, serve to bind sequence-specific DNA binding proteins, i.e., trans-acting factors. The binding of proteins to DNA is responsible for the initiation, maintenance, or down-regulation of gene transcription.
Cis-acting elements which control genes include promoters, enhancers and silencers. Promoters are positioned next to the transcription start site and function in an orientation-dependent manner, while enhancer and silencer elements, which modulate the activity of promoters, may be flexible with respect to their orientation and distance from the transcription start site.
An example of a specifically regulated gene in plants is phenylalanine ammonia-lyase (PAL), which catalyzes the deamination of phenylalanine to cinnamic acid, the precursor of a wide variety of natural products based on the phenylpropane skeleton. During vascular development, PAL is selectively expressed in differentiating xylem cells associated with deposition of the structural polymer lignin. Lignin, the second most abundant biopolymer after cellulose, is the major structural cell wall component of cells forming vessels in plant tissue (xylem). The xylem is responsible for movement of water and inorganic solutes from plant roots to plant shoots. PAL, genes are expressed at correspondingly high levels in differentiating xylem.
The ability to artificially regulate the rate of gene expression provides a means of producing plants with new characteristics. There are numerous situations in which increased levels of gene expression, including increased endogenous gene expression, may be desirable. Such situations include, for example, production of protein plant products for agricultural or commercial purposes.
SUMMARY OF THE INVENTION
The present invention provides a novel repeat element which functions as a non-specific enhancer. In other words, the invention enhancer element does not affect the intrinsic specificity of a promoter associated with the enhancer element. Instead, the enhancer element boosts the activity of the promoter thereby resulting in a desired level of expression of a gene associated with the promoter. A novel transcription factor, palindromic element binding factor (PABF), which binds to the novel repeat element is also provided.
In a first embodiment, the invention provides an enhancer element comprising an isolated nucleotide sequence consisting of at least the sequence (AATT)
n
, where n=2, and preferably from about 2 to about 20. The sequence (AATT)
n
has cis-acting, non-specific, enhancer activity. In one aspect, the invention provides a method for increasing expression of a gene in a cell comprising operably linking a (AATT)
n
repeat element to a heterologous promoter which is operably linked with the gene, thereby permitting increased expression of the gene.
In another embodiment, the invention provides a substantially purified palindromic element binding factor (PABF) polypeptide characterized as having a molecular weight of approximately 67 kDa, as determined by SDS-PAGE, binding to a (AATT)
n
repeat element, where n=2, and having a H1 histone domain, a glutamine rich domain and a high mobility group (HMG) I/Y domain. PABF acts as a transcription factor and binds to the (AATT) repeat element of the invention.


REFERENCES:
Berendsen, H.J.C. (1998) A glimpse of the holy grail? Science. 282:642-643.
Cramer et al. (1989) Phenylalanine ammonia-lyase gene organization and structure. Plant Molecular Biology. 12:367-383.
Tjaden et al. (1994) A novel AT-Rich DNA binding protein that combines an HMG I-like DNA binding domain . . . The Plant Cell. 6:107-118.

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