Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2000-04-10
2001-11-13
McKelvey, Terry (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S091410, C435S243000, C435S320100, C435S325000, C536S023100, C536S023400, C536S023500
Reexamination Certificate
active
06316225
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to novel proteins involved in the initiation of eukaryotic transcription. More specifically, isolated nucleic acid molecules are provided encoding a human Prt1-like subunit protein (hPrt1) and a human eIF4G-like protein (p97). Also provided are hPrt1 and p97 polypeptides, as are vectors, host cells and recombinant methods for producing the same. The invention further relates to screening methods for identifying agonists and antagonists of hPrt1 and p97 activity.
2. Related Art
Eukaryotic protein synthesis requires the participation of translation initiation factors, which assist in the binding of the mRNA to the 40S ribosomal subunit (reviewed in Merrick & Hershey, in
Translational Control
, Hershey et al., eds., Cold Spring Harbour Laboratory Press, (1996), pp. 31-69 and Pain,
Eur. J. Biochem
236:747-771 (1996)). Ribosome binding is facilitated by the cap structure (m
7
GpppN, where N is any nucleotide) that is present at the 5′ end of all cellular mRNAs (except organellar). Biochemical fractionation studies elucidated the general pathway for translation initiation and led to the characterization of several translation initiation factors (reviewed in Merrick & Hershey supra). It is believed that the mRNA cap structure is initially bound by eukaryotic initiation factor (eIF) 4F, which, in conjunction with eIF4B, melts RNA secondary structure in the 5′ untranslated region (UTR) of the mRNA to promote ribosome binding. eIF4F is a more efficient RNA helicase than free eIF4A (Rozen et al.,
Mol. Cell. Biol
. 10:1134-1144 (1990)), consistent with the idea that eIF4A recycles through the eIF4F protein complex to function in unwinding (Pause et al.,
Nature
371:762-767 (1994)). The 40S ribosomal subunit. in a complex with eIF3, eIF1A and eIF2-GTP-tRNAimet, binds at or near the cap structure and scans vectorially the 5′ UTR in search of the initiator AUG codon (reviewed in Merrick & Hershey, supra).
eIF3 is the largest translation initiation factor, with at least 8 different polypeptide subunits and a total mass of approximately 550 to 700 kDa (Schreier, et al.,
J. Mol. Biol
. 116:727-753 (1977); Benne & Hershey,
Proc. Natl. Acad. Sci. USA
73:3005-3009 (1976); Behlke et al
., Eur. J. Biochem
. 157:523-530 (1986)). In mammals, the apparent molecular masses of the eIF3 subunits are 35, 36, 40, 44, 47, 66, 115 and 170 kDa (Behlke, supra; Meyer, et al.,
Biochemistry
21:4206-4212 (1982); Milburn et al.,
Arch. Biochem. Biophys
276:6-11 (1990)). eIF3 is a moderately abundant translation initiation factor, with 0.5 to 1 molecule per ribosome in HeLa cells and rabbit reticulocyte lysates (Meyer, supra; Mengod & Trachsel,
Biochem. Acta
825:169-174 (1985)). This protein complex assumes several functions during translation initiation (reviewed in Hannig,
BioEssays
17:915-919 (1995)). eIF3 binds to the 40S ribosomal subunit and prevents joining with the 60S subunit. It interacts with the ternary complex and stabilizes the binding of the latter to the 40S ribosomal subunit (Trachsel et al.,
J. Mol. Biol
. 116:755-767 (1977); Gupta et al., (1990); Goumans et al.,
Biochem. Biophys. Acta
608:39-46 (1980); Peterson et al.,
J. Biol. Chem
. 254:2509-2510 (1979)). eIF3 crosslinks to mRNA and 18S mRNA (Nygard & Westermann,
Nucl. Acids Res
. 10:1327-1334 (1982); Westermann & Nygard,
Nucl. Acids Res
. 12:8887-8897 (1984)), an activity mainly attributed to the 66 kDa subunit (or 62 kDa in yeast; Garcia-Barrio, et al.,
Genes Dev
. 9:1781-1796 (1995); Naranda, et al.,
J. Biol. Chem
. 269:32286-32292 (1994)). eIF3 co-purifies with eIF4F and eIF4B, two initiation factors involved in the mRNA binding step (Schreier et al.,
J. Mol. Biol
. 116:727-753 (1977)). A direct interaction between the 220 kDa subunit of eIF4F and eIF3 has been demonstrated (Lamphear et al.,
J. Biol. Chem
. 270:21975-21983 (1995)) and a role for eIF3 serving as a bridge between the 40S ribosomal subunit and eIF4F-bound mRNA has been postulated (Lamphear, supra).
The complex structure of eIF3 and its pleiotropic roles in translation initiation have rendered the study of this factor difficult. The protein sequence for only three of the yeast subunits (SUI1/p16, p62 and PRT1/p90) have been published (Garcia-Barrio et al.,
Genes Dev
. 9:1781-1796 (1995); Naranda, supra; Hanic-Joyce et al.,
J. Biol. Chem
. 262:2845-2851 (1987)). However, several other mammalian and yeast subunits have been recently cloned. The yeast protein p90, also known as Prt1, is the most well characterized of those identified to date. Prt1 is an integral subunit of eIF3 (Naranda, supra; Danaie et al.,
J. Biol. Chem
. 270:4288-4292 (1995)). A conditional lethal mutation in the PRT1 gene reduces the binding of the ternary to the 40S ribosomal subunit (Feinberg et al.,
J. Biol. Chem
. 257:10846-10851 (1982)). Other mutations which confer temperature sensitivity are located in the central and carboxy-terminal portion of Prt1. An N-terminal deletion which removes the Prt1 putative RNA Recognition Motif (RRM; for reviews see Birney, et al.,
Nucl. Acids Res
. 21:5803-5816 (1993); Burd & Dreyfuss,
Science
265:615-621 (1994b); Nagai et al.,
Trends Biochem. Sci
. 20:235-240 (1995)), acts a trans-dominant negative inhibitor (Evans et al.,
Mol. Cell. Biol
. 15:4525-4535 (1995)).
Proteins that specifically inhibit cap-dependent translation have been described (Pause, supra; Lin et al.,
Science
266:653-656 (1994)): 4E-binding protein-1 and -2 (4E-BP1 and 4E-BP2) bind to eIF4E and prevent their association with eIF4G, because 4E-BPs and eIF4G share a common site for eIF4E binding (Haghighat et al.,
EMBO J
. 14:5701-5709 (1995); Mader et al.,
Mol. Cell. Biol
. 15:4990-4997 (1995)). Upon treatment of cells with insulin and growth factors, 4E-BPs become phosphorylated. This leads to dissociation of the 4E-BPs from eIF4E and formation of the eIF4F complex, which results in stimulation of translation (Pause, supra; Lin, supra; Beretta, et al.,
EMBO J
. 15:658-664 (1996)).
SUMMARY OF THE INVENTION
The present invention provides isolated nucleic acid molecules comprising polynucleotides encoding the hPrt1 and p97 polypeptides having the amino acid sequences shown in
FIGS. 1A-1D
(SEQ ID NO:2) and
FIGS. 2A-2E
(SEQ ID NO:4) or the amino acid sequences encoded by the cDNA clones deposited as ATCC Deposit Number 97766 on Oct. 18, 1996 and ATCC Deposit Number 97767 on Oct. 18, 1996.
The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and for using them for production of hPrt1 and p97 polypeptides or peptides by recombinant techniques.
The invention further provides isolated hPrt1 and p97 polypeptides having amino acid sequences encoded by polynucleotides described herein.
The present invention also provides a screening method for identifying compounds capable of enhancing or inhibiting a cellular response induced by hPrt1 and/or p97 polypeptides, which involves contacting cells which express hPrt1 and/or p97 polypeptides with the candidate compound, assaying a cellular response, and comparing the cellular response to a standard cellular response, the standard being assayed when contact is made in absence of the candidate compound; whereby, an increased cellular response over the standard indicates that the compound is an agonist and a decreased cellular response over the standard indicates that the compound is an antagonist.
Additional aspects of the invention relate to methods for treating an individual in need of either an increased or decreased level of hPrt1 and/or p97 activity in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of either an isolated hPrt1 and/or p97 polypeptides of the invention (or an agonist thereof) or an hPrt1 and/or p97 antagonist.
The present invention also provides compon
Methot Nathalie
Olsen Henrik Steen
Rom Eran
Ruben Steven Michael
Sonenberg Nahum
Human Genome Sciences Inc.
McKelvey Terry
Sterne Kessler Goldstein & Fox PLLC
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