Abstract: A polypeptide self-antigen useful in a tumor-specific vaccine mimics one or more epitopes of an antigen uniquely expressed by cells of the tumor. The polypeptide is preferably produced in a plant that has been transformed or transfected with nucleic acid encoding the polypeptide and is obtainable from the plant in correctly folded, preferably soluble form without a need for denaturation and renaturation. This plant-produced polypeptide is immunogenic without a need for exogenous adjuvants or other immunostimulatory materials. The polypeptide is preferably an scFv molecule that bears the idiotype of the surface immunoglobulin of a non-Hodgkin's (or B cell) lymphoma. Upon administration to a subject with lymphoma, the plant-produced, tumor-unique scFv polypeptide induces an idiotype-specific antibody or cell-mediated immune response against the lymphoma.

Abstract: A plurality of proteins of interest, or peptides of interest, or other genetically expressed materials, are screened and subsequently produced using any of a variety of expression systems. The plurality of proteins are extracted from a plurality of separate, processed green juices, each green juice containing one of the proteins of interest. A multi-channel apparatus processes the various green juices, one green juice per channel. The apparatus is computer controlled such that the various valves in each channel and pump are controlled in an automated manner to extract each protein of interest and deliver each protein of interest into its own storage vessel.

Abstract: We describe here restriction endonucleases and their uses. Restriction endonucleases are useful in finding single nucleotide polymorphisms. They are also useful in an in vitro method of redistributing sequence variations between non-identical polynucleotide sequences.

Abstract: A polypeptide self-antigen useful in a tumor-specific vaccine mimics one or more epitopes of an antigen uniquely expressed by cells of the tumor. The polypeptide is preferably produced in a plant that has been transformed or transfected with nucleic acid encoding the polypeptide and is obtainable from the plant in correctly folded, preferably soluble form without a need for denaturation and renaturation. This plant-produced polypeptide is immunogenic without a need for exogenous adjuvants or other immunostimulatory materials. The polypeptide is preferably an scFv molecule that bears the idiotype of the surface immunoglobulin of a non-Hodgkin's (or B cell) lymphoma. Upon administration to a subject with lymphoma, the plant-produced, tumor-unique scFv polypeptide induces an idiotype-specific antibody or cell-mediated immune response against the lymphoma.

Abstract: Herein described are various methods for making a vaccine that are made of re-assembled virus like particles (VLP). First, the VLPs are disassembled into coat proteins or encapsidation intermediate populations. Each population undergoes, for instance, chemical conjugation of unique peptide or nucleic moieties to form separate populations. Thereafter, a predetermined amount of each of the several (one or more) different coat proteins or encapsidation intermediates from the different populations is mixed and joined, forming intact VLPs, surrounding a nucleic acid core, that are composed of different coat proteins such that the reassembled VLP displays more than one peptide or other molecule. The nucleic acid can function either as a scaffold alone or can be engineered for the expression of an immunomodulatory protein in a eukaryotic cell.

Abstract: The present invention provides nucleic acid sequences having an altered viral movement protein and 126/183 kDa replicase proteins further characterized in its ability tostabilize a transgene contained in a virus that expresses the altered movement protein. The present invention also provides viral vectors expressing the altered movement protein, cells transformed with the vectors, and host plants infected by the viral vectors.

Abstract: We describe here an in vitro method of increasing complementarity in a heteroduplex polynucleotide sequence. The method uses annealing of opposite strands to form a polynucleotide duplex with mismatches. The heteroduplex polynucleotide is combined with an effective amount of enzymes having strand cleavage activity, 3? to 5? exonuclease activity, and polymerase activity, and allowing sufficient time for the percentage of complementarity to be increased within the heteroduplex. Not all heteroduplex polynucleotides will necessarily have all mismatches resolved to complementarity. The resulting polynucleotide is optionally ligated. Several variant polynucleotides result. At sites where either of the opposite strands has templated recoding in the other strand, the resulting percent complementarity of the heteroduplex polynucleotide sequence is increased. The parent polynucleotides need not be cleaved into fragments prior to annealing heterologous strands. Therefore, no reassembly is required.

Abstract: The present invention provides methods for rapidly determining the function of nucleic acid sequences by transfecting the same into a host organism to effect expression. Phenotypic and biochemical changes produced thereby are then analyzed to ascertain the function of the nucleic acids which have been transfected into the host organism. The invention also provides methods for silencing endogenous genes by transfecting hosts with nucleic acid sequences to effect expression of the same. The present invention also provides methods for selecting desired functions of RNAs and proteins by the use of virus vectors to express libraries of nucleic acid sequence variants. Moreover, the present invention provides methods for inhibiting an endogenous protease of a plant host.

Abstract: A plurality of proteins of interest, or peptides of interest, or other genetically expressed materials, are screened and subsequently produced using any of a variety of expression systems. The plurality of proteins are extracted from a plurality of separate, processed green juices, each green juice containing one of the proteins of interest. A multi-channel apparatus processes the various green juices, one green juice per channel. The apparatus is computer controlled such that the various valves in each channel and pump are controlled in an automated manner to extract each protein of interest and deliver each protein of interest into its own storage vessel.

Abstract: The present invention provides a method for enhancing the production of RNAs or proteins in a plant host using either non-native 5′ untranslated sequences or artificial leader sequences. Preferably, commercially useful proteins, polypeptides, or fusion products thereof are produced, such as, enzymes, antibodies, hormones, pharmaceuticals, vaccines, pigments, anti-microbial polypeptides, and the like. The non-native 5′ untranslated enhancers may also be effective in many different types of transcription or translation systems, such as bacterial and animal systems.

Abstract: We describe here an in vitro method of increasing complementarity in a heteroduplex polynucleotide sequence. The method uses annealing of opposite strands to form a polynucleotide duplex with mismatches. The heteroduplex polynucleotide is combined with an effective amount of enzymes having strand cleavage activity, 3′ to 5′ exonuclease activity, and polymerase activity, and allowing sufficient time for the percentage of complementarity to be increased within the heteroduplex. Not all heteroduplex polynucleotides will necessarily have all mismatches resolved to complementarity. The resulting polynucleotide is optionally ligated. Several variant polynucleotides result. At sites where either of the opposite strands has templated recoding in the other strand, the resulting percent complementarity of the heteroduplex polynucleotide sequence is increased. The parent polynucleotides need not be cleaved into fragments prior to annealing heterologous strands. Therefore, no reassembly is required.

Abstract: Restriction Independent Cloning Events (RICE) are made by generating 5′ overhangs (sticky ends). The polynucleotides to be joined are reacted with a DNA polymerase, having 3′ to 5′ exonuclease activity and 5′ to 3′ polymerizing activity, less than all of the dNTPs, a kinase (optional) and a ligase. The complementary 5′ overhangs anneal and ligate. Further disclosed is a method of joining double stranded polynucleotides that includes joining a first DNA sequence and a second DNA sequence using topoisomerase I to form a first product, joining the second DNA sequence and a third DNA sequence using topoisomerase I to form a second product, and combining the first product and the second product in PCR reaction to generate a third product.

Abstract: We describe here an in vitro method of redistributing sequence variations between non-identical polynucleotide sequences, by making a heteroduplex polynucleotide from two non-identical polynucleotides; introducing a nick in one strand at or near a base pair mismatch site; removing mismatched base(s) from the mismatch site where the nick occurred; and using the opposite strand as template to replace the removed base(s) with bases that complement base(s) in the first strand. By this method, information is transferred from one strand to the other at sites of mismatch.

Abstract: We describe here an in vitro method of redistributing sequence variations between non-identical polynucleotide sequences, by making a heteroduplex polynucleotide from two non-identical polynucleotides; introducing a nick in one strand at or near a base pair mismatch site; removing mismatched base(s) from the mismatch site where the nick occurred; and using the opposite strand as template to replace the removed base(s) with bases that complement base(s) in the first strand. By this method, information is transferred from one strand to the other at sites of mismatch.

Abstract: The present invention relates to foreign peptide sequences fused to recombinant plant viral structural proteins and a method of their production. Fusion proteins are economically synthesized in plants at high levels by biologically contained tobamoviruses. The fusion proteins of the invention have are useful as antigens for inducing the production of antibodies having desired binding properties, e.g., protective antibodies, or for use as vaccine antigens for the induction of protective immunity against the parvovirus. Feline parvovirus epitopes were fused to the N-terminus of the TMV coat protein, expressed in Nicotiana plants, extracted, purified, characterized and administered to animals, resulting in protective immunity.

Abstract: Herein described are various methods for making a vaccine that are made of re-assembled virus like particles (VLP). First, the VLPs are disassembled into encapsidation intermediate populations. Each encapsidation intermediate population undergoes, for instance, chemical conjugation of unique peptide or nucleic moieties to form separate populations. Thereafter, a predetermined amount of each of the several (one or more) different encapsidation intermediates from the different populations is mixed and joined, forming intact VLPs, surrounding a nucleic acid core, that are composed of different encapsidation intermediate such that the reassembled VLP displays more than one peptide or nucleic acid. The nucleic acid can function either as a scaffold alone or can be engineered for the expression of an immunomodulatory protein in a eukaryotic cell.

Abstract: The present invention provides nucleic acid sequences having an altered viral movement protein and 126/183 kDa replicase proteins further characterized in its ability to stabilize a transgene contained in a virus that expresses the altered movement protein. The present invention also provides viral vectors expressing the altered movement protein, cells transformed with the vectors, and host plants infected by the viral vectors.

Abstract: Restriction Independent Cloning Events (RICE) are made by generating 5′ overhangs (sticky ends). The polynucleotides to be joined are reacted with a DNA polymerase, having 3′ to 5′ exonuclease activity and 5′ to 3′ polymerizing activity, less than all of the dNTPs, a kinase (optional) and a ligase. The complementary 5′ overhangs anneal and ligate. Further disclosed is a method of joining double stranded polynucleotides that includes joining a first DNA sequence and a second DNA sequence using topoisomerase I to form a first product, joining the second DNA sequence and a third DNA sequence using topoisomerase I to form a second product, and combining the first product and the second product in PCR reaction to generate a third product.

Abstract: Restriction Independent Cloning Events (RICE) are made by generating 5′ overhangs (sticky ends). The polynucleotides to be joined are reacted with a DNA polymerase, having 3′ to 5′ exonuclease activity and 5′ to 3′ polymerizing activity, less than all of the dNTPs, a kinase (optional) and a ligase. The complementary 5′ overhangs anneal and ligate.

Abstract: We describe here an in vitro method of increasing complementarity in a heteroduplex polynucleotide sequence. The method uses annealing of opposite strands to form a polynucleotide duplex with mismatches. The heteroduplex polynucleotide is combined with an effective amount of enzymes having strand cleavage activity, 3′ to 5′ exonuclease activity, and polymerase activity, and allowing sufficient time for the percentage of complementarity to be increased within the heteroduplex. Not all heteroduplex polynucleotides will necessarily have all mismatches resolved to complementarity. The resulting polynucleotide is optionally ligated. Several variant polynucleotides result. At sites where either of the opposite strands has templated recoding in the other strand, the resulting percent complementarity of the heteroduplex polynucleotide sequence is increased. The parent polynucleotides need not be cleaved into fragments prior to annealing heterologous strands. Therefore, no reassembly is required.