Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-07-21
2002-10-22
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S069100, C435S091400, C435S410000, C435S005000, C435S468000, C435S440000, C435S235100, C435S441000, C435S446000, C536S023100
Reexamination Certificate
active
06468745
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to the field of molecular biology and viral genetics. Specifically, the present invention relates to a method for using viral vectors to house populations of nucleic acid sequence variants and to select one or more sequences that exhibit the desired traits using plant hosts.
BACKGROUND OF THE INVENTION
Nature has developed many strategies for generating genetic diversity over billions of years of evolution. These strategies include random mutagenesis, recombination and selection. Many methods are now available in the laboratory to mimic these processes in order to efficiently generate beneficial mutations and select molecules with desired traits. For recent reviews, see Minshull et al.,
Curr. Opin. Chem. Biol.
3:284-290 (1999); Giver et al., ibid 2:335-338 (1998); and Patten et al., ibid 8:724-733 (1997).
The generation of genetic diversity through in vitro recombination methods is often referred to as “molecular breeding” or “directed evolution” (Minshull et al., supra and Kuchner et al.,
Trends Biotechnol.
15:523-530 (1997)). DNA shuffling is a method for generating, in vitro, recombinant genes from a set of parent genes (Stemmer,
Nature,
370:389-391 (1994); Stemmer,
Proc. Natl. Acad. Sci. USA
91:10747-10751 (1994); Crameri et al.,
Nat. Biotechnol.
14:315-319 (1996); Crameri et al.,
Nature Medicine
2:100-103 (1996); Stemmer,
Sexual PCR and Assembly PCR
in
The Encyclopedia of molecular Biology,
VCH Publishers, New York, pp. 447-457 (1996); Crameri et al.,
Nat. Biotechnol.
15:436-438 (1997); Zhang et al.,
Proc. Natl. Acad. Sci. USA
94:4504-4509 (1997); Crameri et al.,
Nature
391:288-291 (1998); Christians et al.,
Nat. Biotechnol.
17:259-264 (1999), U.S. Pat. Nos. 5,830,721, 5,811,238, 5,830,721, 5,605,793, 5,834,252, and 5,837,458; and PCT publications WO 98/13487, WO98/27230, and WO 98/31837). Typically, the parental genes are randomly fragmented by Dnase I. The purified fragments are then reassembled by repeated cycles of overlap extension into full-length genes that contain novel combinations of the parental mutations.
Other in vitro recombination methods have also been developed to generate a population of nucleic acid sequences, for example, random priming recombination (RPR) and the staggered extension process (StEP) (Moore et al.,
Nat. Biotechnol.
14:458-467 (1996); Zhao et al.,
Proc. Natl. Acad. Sci. USA
94:7997-8000 (1997); Arnold,
Acc. Chem. Res.
31:125-131 (1998); Shao et al.,
Nucleic Acids Res.
26:681-683 (1998); Zhao et al.,
Nat. Biotechnol.
16:258-261 (1998); Arnold,
Proc. Natl. Acad. Sci. USA
95:2035-2036 (1998); Giver et al.,
Proc. Natl. Acad. Sci. USA
95:12809-12813 (1998); and Zhao et al.,
Protein Eng.
12:47-53 (1999)). In the RPR method, short random primers are annealed to the template and extended by polymerase. The resulting fragments, the length of which can be controlled by altering the conditions of the annealing and extension reaction, are then separated from the initial template and unextended primers. These fragments are assembled into full length genes by cycles of overlap extension. The StEP method uses template switching during synthesis to form the desired chimeric genes. The templates are mixed with one or more primers and subjected to repeated cycles of denaturation and short annealing/extension steps. Because the growing fragments can anneal to different templates, the resulting full length sequences contain sequence information from different parents.
DNA shuffling and other in vitro recombination methods have been applied to prokaryotic or cell-base systems to select sequences of desired protein activities. However, the ability to introduce sequence variants throughout an organism in a rapid and high throughput manner has not been demonstrated. Virus vectors are ideal for shuttling libraries of sequence variants throughout an organism, such as plants, for selection of optimized functions. No other tool, transient or stable expression methods, can match the ability of viral vectors to develop optimized functions using plant hosts.
Viruses are a unique class of infectious agents whose distinctive features are their simple organization and their mechanism of replication. Their hosts include a wide variety of plants and animals. A complete viral particle, or virion, may be regarded mainly as a block of genetic material (either DNA or RNA) capable of autonomous replication, surrounded by a protein coat and sometimes by an additional membranous envelope. The coat protects the virus from the environment and serves as a vehicle for transmission from one host cell to another.
Foreign genes can be expressed in plant hosts either by permanent insertion into the genome or by transient expression using virus-based vectors. Each approach has its own distinct advantages. Transformation for permanent expression needs to be done only once, whereas each generation of plants needs to be inoculated with the transient expression vector. Virus-based expression systems, in which the foreign mRNA is greatly amplified by virus replication, can produce very high levels of proteins in leaves and other tissues. Viral vector-produced protein can also be directed to specific subcellular locations, such as endomembrane, cytosol, or organelles, or it can be attached to macromolecules, such as virions, which aids purification of the protein. For the production of some products, including products for the human health industry, plants provide an optimal system because of reduced capital costs and the greater potential for large-scale production compared with microbial or animal systems.
In this invention, we describe the use of viral expression vectors to bear populations of sequence variants. Plant hosts are used to select those sequences with desired properties, which may be further characterized.
SUMMARY OF THE INVENTION
The present invention is a method for selecting desired traits in a plant host by the use of viral vectors to express libraries of nucleic acid sequence variants. This in vitro evolution method is used to improve virus-specific, protein-specific, or host-specific functions. Libraries of sequence variants may be generated by in vitro mutagenesis and/or recombination methods, such as chemical treatment, oligonucleotide mediated mutagenesis, PCR mutagenesis, DNA shuffling, random priming recombination (RPR), restriction enzyme fragment induced template switching (REFITS), and the staggered extension process (StEP), among others. Libraries of sequence variants may be random, semi-random or known sequences. In preferred embodiments, RNA viral vectors may be used as the genetic backbones to bear libraries containing variants of nucleic acid sequences and to be applied to plant hosts such that the desired traits in the RNA or protein products can be determined, selected and improved. The template nucleic acid sequences for generating sequence variants may be of viral origin, such as, sequences encoding, coat protein, movement protein, promoter, internal initiation sites, packaging signals, 5′ and 3′ NTRs, or ribosomal sequences, or any other structural and non-structural components of viral nucleic acid sequences. The template nucleic acid sequences for generating sequence variants may also be derived from genes, regulatory sequences, or fragments thereof from bacteria, fungi, plants, animals or other sources. These non-native sequences may be inserted in viral vectors to express foreign proteins, regulate transcription or translation, increase the genetic stability of foreign sequences in viral vectors, etc.
After a plant host is infected with a library containing populations of sequence variants, one or more desired traits are screened and selected. The desired traits may include biochemical or phenotypic traits. Phenotypic traits may include, but not limited to, host range, viral infectivity, tolerance to herbicides, tolerance to extremes of heat or cold, drought, salinity or osmotic stress; resistance to pests (insects, nematodes or arachnids)
Fitzmaurice Wayne P.
Lindbo John A.
Padgett Hal S.
Pogue Gregory P.
Chiang Robin C.
Gallegos Thomas
Guzo David
Halluin Albert P.
Large Scale Biology Corporation
LandOfFree
Method for expressing a library of nucleic acid sequence... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method for expressing a library of nucleic acid sequence..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method for expressing a library of nucleic acid sequence... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2991139