Factors involved in gene expression

Details Factors involved in gene expression Factors involved in gene expression

1999-07-02

2001-09-25

Carlson, Karen Cochrane (Department: 1653)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S023100, C536S023600, C435S320100, C435S252300, C435S419000, C435S254200

Reexamination Certificate

active

06294658

ABSTRACT:

FIELD OF THE INVENTION
This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding factors involved in gene expression in plants and seeds.
BACKGROUND OF THE INVENTION
Binding of 40S ribosomal subunit to mRNA requires several initiation factors, including proteins that recognize the cap structure. In the initiation of translation in eukaryotes, binding of the small ribosomal subunit to the mRNA results from recognition of the 5′ cap structure of the mRNA by the cap-binding complex eukaryotic initiation factor 4F (eIF4F). This factor is itself a three-subunit complex comprising the cap binding protein eIF4E, eIF4A, an ATP-dependent RNA helicase, and eIF-4 gamma, which interacts with both eIF4E and eIF4A and enhances cap binding by eIF4E. Eukaryotic protein synthesis initiation factor eIF-4 gamma, also known as p220, is involved in the recognition of the mRNA cap, ATP-dependent unwinding of 5′-terminal secondary structure and recruitment of mRNA to the ribosome.
Infection of mammalian cells by picornaviruses results in the proteolytic cleavage of eIF-4 gamma, severely restricting cap-dependent translation initiation but permitting cap-independent initiation to proceed from an internal ribosome entry site. Processing of eIF-4 gamma by potyviruses (plant members of the picornavirus superfamily) has not been shown to date but this process may also occur in plants.
eIF4F activity is impaired during heat shock, when binding of eIF4E to its inhibitory protein (4E-BP1) is increased and the amount of eIF-4 gamma is decreased. Heat shock protein mRNAs are believed to be relatively cap-independent, providing a mechanism for the selective up-regulation of the synthesis of heat shock proteins and other stress proteins during heat shock (Vries et al. (1997)
J. Biol. Chem.
272: 32779-32784). The eIF-4 gamma polypeptide from rabbit or human is 154 kDa (1,396 amino acid residues) and contains sequence motifs of potential interest: a sequence (AGLGPR) that is similar to the substrate recognition sequence of protease 2A from rhinovirus serotype 14, five PEST regions with scores greater than 10 (which are characteristic of rapidly degraded proteins), stretches of polyglutamic acid, and numerous potential phosphorylation sites (Rychlik et al. (1992)
J. Biol. Chem.
267: 23226-23231). Sequences encoding eIF-4 gamma have been studied in humans, mammals, and yeast.
The mRNA 3′ poly(A) tail and the associated poly(A)-binding protein also regulate translational initiation, probably by interacting with the 5′ end of the mRNA (Craig et al. (1998)
Nature
392:520-523). The poly(A) binding protein is essential for viability of the yeast
Saccharomyces cerevisiae.
The amino acid sequence of the protein indicates that it consists of four ribonucleoprotein consensus sequence-containing RNA-binding domains and a proline-rich auxiliary domain at the carboxyl terminus. Specific poly(A) binding activity was found only in the two amino-terminal RNA-binding domains which, interestingly, are dispensable for viability of yeast cells (Burd et al. (1991)
Mol. Cell Biol.
11: 3419-3424). Multiple poly(A) binding protein-related sequences have been cloned from
Arabidopsis thaliana
suggesting that this protein is encoded by a multigene family.
SUMMARY OF THE INVENTION
The instant invention relates to isolated nucleic acid fragments encoding factors involved in gene expression. Specifically, this invention concerns an isolated nucleic acid fragment encoding a poly(A) binding protein or a eukaryotic translation initiation factor-4 gamma (eIF-4 gamma) and an isolated nucleic acid fragment that is substantially similar to an isolated nucleic acid fragment encoding a poly(A) binding protein or a eIF-4 gamma. In addition, this invention relates to a nucleic acid fragment that is complementary to the nucleic acid fragment encoding poly(A) binding protein or eIF-4 gamma.
An additional embodiment of the instant invention pertains to a polypeptide encoding all or a substantial portion of a factor involved in gene expression selected from the group consisting of poly(A) binding protein and eIF-4 gamma.
In another embodiment, the instant invention relates to a chimeric gene encoding a poly(A) binding protein or a eIF-4 gamma, or to a chimeric gene that comprises a nucleic acid fragment that is complementary to a nucleic acid fragment encoding a poly(A) binding protein or a eIF-4 gamma, operably linked to suitable regulatory sequences, wherein expression of the chimeric gene results in production of levels of the encoded protein in a transformed host cell that is altered (i.e., increased or decreased) from the level produced in an untransformed host cell.
In a further embodiment, the instant invention concerns a transformed host cell comprising in its genome a chimeric gene encoding a poly(A) binding protein or a eIF-4 gamma, operably linked to suitable regulatory sequences. Expression of the chimeric gene results in production of altered levels of the encoded protein in the transformed host cell. The transformed host cell can be of eukaryotic or prokaryotic origin, and include cells derived from higher plants and microorganisms. The invention also includes transformed plants that arise from transformed host cells of higher plants, and seeds derived from such transformed plants.
An additional embodiment of the instant invention concerns a method of altering the level of expression of a poly(A) binding protein or a eIF-4 gamma in a transformed host cell comprising: a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding a poly(A) binding protein or a eIF-4 gamma; and b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of altered levels of poly(A) binding protein or eIF-4 gamma in the transformed host cell.
An additional embodiment of the instant invention concerns a method for obtaining a nucleic acid fragment encoding all or a substantial portion of an amino acid sequence encoding a poly(A) binding protein or a eIF-4 gamma.
A further embodiment of the instant invention is a method for evaluating at least one compound for its ability to inhibit the activity of a poly(A) binding protein or a eIF-4 gamma, the method comprising the steps of: (a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding a poly(A) binding protein or a eIF-4 gamma, operably linked to suitable regulatory sequences; (b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of poly(A) binding protein or eIF-4 gamma in the transformed host cell; (c) optionally purifying the poly(A) binding protein or the eIF-4 gamma expressed by the transformed host cell; (d) treating the poly(A) binding protein or the eIF-4 gamma with a compound to be tested; and (e) comparing the activity of the poly(A) binding protein or the eIF-4 gamma that has been treated with a test compound to the activity of an untreated poly(A) binding protein or eIF-4 gamma, thereby selecting compounds with potential for inhibitory activity.


REFERENCES:
GenEmbl Accession No. P93616. Le et al. Poly (A) binding protein, May 1, 1997.*
Watson et al. Recombinant DNA, 2nd Edition. pp. 453-455, 1992.*
Vries et al., (1997) J. Biol. Chem., 272:32779-32784.
Yam et al., (1992) J. Biol. Chem. 267:23226-23231.
Craig et al., (1998), Nature 392:520-523.
Burd et al., (1991) Mol. Cell biol. 11:3419-3424.
Mol. Cell Biol. 17(12), 6940-6947 (1997) Imataka and Sonenberg.
J. Biol. Chem. 268(26), 19200-19203, (1993) Lamphear et al.

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