Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-06-23
2002-09-10
Horlick, Kenneth R. (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S252100, C435S320100, C536S023100
Reexamination Certificate
active
06448007
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the field of nucleic acid regulatory elements that affect mRNA translation, export, and stability. More specifically, the invention relates to the screening of 5′ and 3′ untranslated RNA sequences, the identification of RNA regulatory elements within these sequences, and the identification of compounds that modulate the function of these RNA regulatory sequences.
BACKGROUND
While transcriptional controls regulate gene expression by influencing the rate of mRNA production, post-transcriptional mechanisms can also regulate gene expression by modulating the amount of protein produced from an mRNA molecule. For example, gene expression can be regulated by altering mRNA translation efficiency (Izquierdo and Cueza, Mol. Cell Biol. 17: 5255-5268, 1997; Yang et al., J. Biol. Chem. 272: 15466-73, 1997), or by altering mRNA stability (Ross, Microbiol. Rev. 59: 423-50, 1995). Post-transcriptional control mechanisms appear to play an especially important role in the gene expression response to environmental factors, such 1s response to heat shock (Sierra et al., Mol. Biol. Rep. 19: 211-20, 1994), iron availability (Hentze et al., Proc. Natl. Acad.
Sci. USA 93: 8175-82, 1996), oxygen availability (Levy et al., J. Biol. Chem. 271: 2746-53, 1996; McGary et al., J. Biol. Chem. 272: 8628-34, 1997), and growth factors (Amara et al., Nucleic Acids Res. 21: 4803-09, 1993).
Post-transcriptional regulatory elements may be present in the 5′ and 3′ mRNA untranslated regions (UTRs). At the 5′ UTR, mRNA binding to ribosomes is generally the rate-limiting step in translation initiation (Mathew et al., In: Translational Control, pages 1-30, Eds: Hershey et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1996). At the 3′ UTR, regulatory elements may modulate mRNA translation and degradation, as well as mRNA transport and subcellular localization (Jackson, Cell 74: 9-14, 1993). However, the nature of most UTR post-transcriptional elements remains poorly understood. A method for efficiently characterizing these mRNA regulatory sequences would advance the discovery of compounds that modulate expression of therapeutically important proteins via regulatory mRNA sites.
SUMMARY OF THE INVENTION
We have discovered a method for constructing libraries that are specifically biased for RNA regulatory sites. In the first aspect, the invention features a cDNA library consisting essentially of at least 100 different cDNA sequences that correspond to different mRNA untranslated region (UTR) sequences isolated and separate from adjacent mRNA coding sequences. Preferably, the cDNA sequences are cloned into a vector system that can express the sequences, and such a vector is also a feature of this invention. This vector includes the following: a) a nucleotide sequence encoding an mRNA UTR sequence in operative linkage to a promoter, wherein the nucleotide sequence is derived from the cDNA library of the first aspect; b) a first reporter gene positioned for transcription upstream or downstream of the UTR-encoding nucleotide sequence; and c) a second, different reporter gene in operative linkage to a promoter but unassociated with the UTR-encoding nucleotide sequence. Preferably, the reporter genes encode a fluorescent protein or cell surface marker protein.
A second and related aspect of the invention features a cDNA library, wherein the library is constructed by steps that include the following: a) purifying poly(A)+ RNA from total RNA; b) performing controlled, non-random enzymatic digestion of AUG sequences in the poly(A)+ RNA; c) purifying the digested RNA to obtain the fragments containing the 5′ end sequences; and d) synthesizing cDNA from the purified RNA obtained in step (c); wherein the library consists essentially of cDNA sequences corresponding to mRNA 5′ untranslated region (UTR) sequences, isolated and separate from adjacent mRNA coding sequences. Preferably, the enzymatic digestion is carried out using RNase H.
In a third aspect, the invention features a cDNA library constructed by steps that include the following: a) purifying poly(A)+ RNA from total RNA; b) synthesizing nucleic acid heteroduplexes from the poly(A)+ RNA using degenerate primers that hybridize preferentially to the region surrounding and including the initiation codon, where the heteroduplexes comprises the 5′ end sequences of the RNA; c) purifying the heteroduplexes obtained in step (b) to obtain the fragments containing the 5′ end sequences; and d) synthesizing cDNA from the purified heteroduplexes obtained in step (c); wherein the library consists essentially of cDNA sequences corresponding to mRNA 5′ untranslated (UTR) sequences, isolated and separate from adjacent mRNA coding sequences.
In one embodiment of any of the above three aspects of the invention, the cDNA library consists essentially of cDNA sequences corresponding to mRNA untranslated region sequences, isolated in intact form.
In preferred embodiments of the second or third aspects of the invention, the 5′ sequence purification is carried out using a cap binding protein, for example, an eIF4E fusion protein or an antibody to the 5′ cap, and the DNA sequences are cloned into a vector system that can express the sequences. This vector includes the following: a) a nucleotide sequence encoding an mRNA UTR sequence in operative linkage to a promoter, wherein the nucleotide sequence is derived from the cDNA library of the second or third aspect; b) a first reporter gene positioned for transcription upstream or downstream of the UTR-encoding nucleotide sequence; and c) a second, different reporter gene in operative linkage to a promoter but unassociated with the UTR-encoding nucleotide sequence. Preferably, the reporter genes encode a fluorescent protein or cell surface marker protein.
A related fourth aspect of the invention is a cDNA library, wherein the library is constructed by steps that include the following: a) purifying poly(A)+ RNA from total RNA; b) performing random digestion on the poly(A )+ RNA; c) purifying the digested RNA to obtain poly(A) containing fragments; and d) synthesizing cDNA from the purified RNA obtained in step (c); wherein the library consists essentially of cDNA sequences corresponding to 3′ UTR sequences, isolated and separate from adjacent mRNA coding sequences.
A cDNA library is also featured in the fifth aspect of the invention. This cDNA library is constructed by steps that include the following: a) purifying poly(A)+ RNA from total RNA; b) loading the poly(A)+ RNA with ribosomes; and c) performing reverse transcription on the loaded poly(A)+ RNA using an oligo(dT) primer and polymerase; wherein the library consists essentially of cDNA sequences corresponding to 3′ UTR sequences, isolated and separate from adjacent mRNA coding sequences. Preferably, the cDNA sequences of the libraries of the fourth or fifth aspects are cloned into vector systems that can express the sequences, and such vectors are also a feature of this invention. These vectors include the following: a) a nucleotide sequence encoding an mRNA UTR sequence in operative linkage to a promoter, wherein the nucleotide sequence is derived from the cDNA library of the fourth or fifth aspect; b) a first reporter gene positioned for transcription upstream or downstream of the UTR-encoding nucleotide sequence; and c) a second, different reporter gene in operative linkage to a promoter but unassociated with the UTR-encoding nucleotide sequence. Preferably, the reporter genes encode a fluorescent protein or cell surface marker protein.
In one embodiment of the fourth or fifth aspect the invention, the cDNA library consists essentially of cDNA sequences corresponding to 3′ untranslated region sequences, isolated in intact form.
A sixth aspect of the invention provides a method of identifying a regulatory UTR sequence that includes the following steps: a) transfecting a plurality of host cell
Burnett Bonnie-Ann
Eder Paul S.
Giordano Tony
Powers Gordon D.
Temeles Gretchen L.
Clark & Elbing LLP
Horlick Kenneth R.
Message Pharmaceuticals
Strzelecka Teresa
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