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
1997-03-05
2001-04-17
Carlson, Karen Cochrane (Department: 1653)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S320100, C435S252300, C435S419000, C435S410000, C435S411000, C536S023100, C536S023200, C536S023600
Reexamination Certificate
active
06218142
ABSTRACT:
SUMMARY OF THE INVENTION
The present invention relates to nucleic acid molecules encoding polypeptides having the enzymatic activity of an RNA-directed RNA polymerase (RdRP). The present invention also provides vectors comprising said nucleic acid molecules, wherein the nucleic acid molecules are operatively linked to regulatory elements allowing expression in prokaryotic and/or eukaryotic host cells. Additionally, the present invention relates to polypeptides encoded by said nucleic acid molecules and methods for the production of said polypeptides. The present invention further relates to pharmaceutical and diagnostic compositions as well as kits comprising the aforementioned nucleic acid molecules and/or comprising a nucleic acid molecule which is complementary to such a nucleic acid molecule. Said compositions and kits may further comprise polypeptides encoded by the described nucleic acid molecules. Furthermore, the present invention relates to antagonists and inhibitors of the aforesaid polypeptides and/or antibodies specifically recognizing such polypeptides. Also within the scope of the present invention are methods and uses comprising the nucleic acid molecules, vectors, polypeptides, antibodies and antagonists and inhibitors of the invention for modulating gene expression in humans and animals. Furthermore, the present invention relates to transgenic plant cells and plants containing the aforementioned nucleic acid molecules as well as the use of the aforementioned nucleic acid molecules, polypeptides and/or antagonists/inhibitors in plant cell culture and plant tissue culture and/or plant breeding.
BACKGROUND OF THE INVENTION
Since the development of plant transformation systems a particular interest exists on the stable expression of foreign genes in higher plants. Although these attempts were successfully realized for several herbicide resistance genes, a series of experiments failed that had been designed to use plants as efficient bio-reactors. It seemed that higher plants have a system of defense to protect themselves against overexpression of foreign genes. Whenever a transgene expression comes up to a threshold dose it is without selective pressure silenced either by transcriptional or post-transcriptional inactivation. Recent studies on these phenomena indicated that “antisense” RNA might be responsible for these silencing mechanisms. The idea of an “antisense” RNA-mediated gene silencing was substantiated by the analysis of transgenically mediated virus resistance in plants (Smith et al., 1994: English et al., 1996; Sijen et al., 1996). It could be shown that the in vivo transcripts of non-translatable “sense” cDNA constructs which had been integrated into the plant genome can mediate resistance against infection with plant viruses. Further analysis revealed that this resistance which was due to post-transcriptional gene silencing (PTGS) only occurred when the transgene shared homology with the infecting virus. Based on these data and with regard to previous results describing the co-suppression phenomena in plants (for review see Meyer, 1996) several models explaining the PTGS mechanism have been introduced (English et al., 1996; Baulcomb, 1996; Sijen et al., 1998). Common to all these models is the assumption that “antisense” RNAs are synthesized from “sense” RNA templates by an RNA-directed RNA polymerase (RdRP). Subsequently the produced “antisense” RNAs can hybridize to complementary parts of mRNAs or invading virus RNAs which would unresistingly lead to degradation of the double-stranded regions.
It is most likely that this PTGS is not restricted to transgenes but that it is also involved in normal plant gene regulation. Moreover, it is most likely that the same mechanism takes place in all higher eukaryots. Therefore, a detailed examination of RNA-mediated gene regulation had become an important aspect of basic and applied research. An RNA-dependent RNA polymerase (EC 2.7.7.48, RdRP) activity has been detected in healthy plant tissue, for review see Fraenkel-Conrat (1986). Previous studies of RdRP activity have suffered from the inhomogeneity of enzyme preparations and of the resulting RNA products in that they did not allow the precise determination of their template complementarity by direct RNA sequencing. Schiebel et al. (1993 a, b) provided evidence for the concept that the RdRP mediated transcription is a truly RNA-instructed process yielding products that are precise complementary copies of the RNA template offered to an RdRP active enzyme preparation. However, although purification of the RdRP from tomato leaf tissue to electrophoretic homogeneity was reported (Schiebel et al., 1993 a, b), the enzyme preparations were not approachable for amino acid sequencing. One reason for the slow progress in elucidating the amino acid sequence of the RdRP is to be seen in the fact that only low amounts of protein could be isolated using the hitherto available technology and/or that there existed further proteins in the enzyme preparations obtainable with standard techniques which resulted in non-informative or even false sequence information. However, for efficient use of RdRP associated technology, it was desirable to be able to manipulate the genetic material associated with said technology.
Thus, the technical problem underlying the present invention is to provide such material.
DETAILED DESCRIPTION OF THE INVENTION
The solution to the technical problem is achieved by providing the embodiments characterized in the claims.
Accordingly, the invention relates to a nucleic acid molecule encoding a polypeptide having the enzymatic activity of an RNA-directed RNA polymerase (RdRP) or encoding an enzymatically active fragment thereof selected from the group consisting of:
(a) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence given in SEQ ID NO:2;
(b) nucleic acid molecules comprising the nucleotide sequence given in SEQ ID NO:1;
(c) nucleic acid molecules hybridizing with a complementary strand of a nucleic acid molecule as defined in (a) or (b); and
(d) nucleic acid molecules, the nucleotide sequence of which is degenerate as a result of the genetic code to a nucleotide sequence of a nucleic acid molecule as defined in (c).
In accordance with the present invention, a nucleic acid molecule encoding a novel class of RdRPs has been identified. This has been achieved by using a novel purification method for the isolation of a polypeptide having the enzymatic activity of an RdRP which was suitable for amino acid sequencing. Oligonucleotides were designed and used for cloning of the corresponding cDNA.
The polypeptide encoded by the nucleic acid sequence of SEQ ID NO: 1 from nucleotide 194 to nucleotide 3535 encodes a polypeptide of a 1114 amino acids with a calculated molecular weight of about 127 kDa. This data is in good agreement with the prior art experimental values that had been determined by SDS-PAGE (128 kD) and sucrose gradient centrifugation (119 kD) (Schiebel et al., 1993a). The nucleotide and the amino acid sequence given in SEQ ID NOS: 1 and 2, respectively, displayed no significant homologies to sequences in any database (Blast X, GAP, Wisconsin Sequence Packaging System, Version 7.0) searched. In addition, the presence of putative phosphorylation sites for putative protein kinases can be identified by computer aided search for phosphorylation consensus sequences which suggests possible regulation by phosphorylation. Studies which had been carried out in accordance with the present invention revealed that the polypeptide encoded by the nucleotide sequence of SEQ ID NO:1 is identical with the RdRP isolated from tomato plants as described in example 1 of the present application.
The polypeptide encoded by the nucleotide sequence of SEQ ID NO:1 displays the ability to catalyze in vitro transcription of preferably short single-stranded RNA and DNA molecules into precisely complementary RNA copies up to the full length of these templates. The RdRP-directed transcription can be primed by RNA and DNA oligonucleotides comple
Riedel Leonhard
Sanger Heinz L.
Schiebel Winfried
Wassenegger Michael
Brown Karen E.
Carlson Karen Cochrane
Fish & Neave
Haley Jr. James F.
Schnizer Holly
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