Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2000-11-09
2003-03-11
Whisenant, Ethan C. (Department: 1655)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S006120, C435S091100, C435S440000, C435S463000
Reexamination Certificate
active
06531316
ABSTRACT:
FIELD OF THE INVENTION
The present invention provides methods of encrypting traits, including, e.g., splitting genes between two parental organisms or between a host organism and a vector. The invention also relates to methods of unencrypting trait encrypted gene sequences to provide unencrypted RNAs or polypeptides. Gene sequences are unencrypted when the two parental organisms are mated, or when the vector infects the host organism by trans-splicing either the split RNAs, or the split polypeptides upon expression of the split gene sequences. The invention also includes methods of providing multiple levels of trait encryption and reliable methods of producing hybrid organisms. Additional methods include those directed at unencrypting engineered genetic elements to provide unencrypted polypeptide functions and those related to recombining non-overlapping gene sequences. Furthermore, the present invention includes integrated systems and various compositions related to the methods disclosed herein.
BACKGROUND OF THE INVENTION
Intermolecular splicing is termed trans-splicing. The mechanism of splicing two independently transcribed pre-mRNAs was discovered in trypanosomes. Murphy, W. J. et al. (1986)
Cell
47, 517-525 and Sutton, R. and Boothroyd, J. C. (1986)
Cell
47, 527-535. Thereafter, trans-splicing was also described in other organisms, e.g.,
C. elegans
(Krause, M. and Hirsch, D. (1987)
Cell
49, 753-761, Huang, X. Y. and Hirsch, D. (1989)
Proc. Nat. Acad. Sci. USA
86, 8640-8644, and Hannon, G. J. et al. (1990)
Cell
61, 1247-1255),
Schistosoma mansoni
(Rajkovic, A., et al. (1990)
Proc. Nat. Acad. Sci. USA
87, 8879-8883 and Davis, R. E. et al. (1995)
J. Biol. Chem.
270, 21813-21819), and plant mitochondria (Malek, O. et al. (1997)
Proc. Nat. Acad. Sci. USA
94, 553-558). Targeted trans-splicing has been demonstrated in HeLa nuclear extracts, in cultured H1299 human lung cancer cells, and in H1299 tumor bearing athymic mice. Puttaraju, M. et al. (1999)
Nat. Biotech.
17, 246-252. Suggested practical applications of targeted trans-splicing are, e.g., as a means for gene therapy. Id.
Various ribozymes capable of precisely trans-splicing, either in vitro or in vivo, exon sequences into target RNA sequences have been described in, e.g., Haseloff et al., U.S. Pat. No. 5,882,907 “CELL ABLATION USING TRANS-SPLICING RIBOZYMES,” Haseloff et al., U.S. Pat. No. 5,874,414 “TRANS-SPLICING RIBOZYMES,” Haseloff et al., U.S. Pat. No. 5,866,384 “CELL ABLATION USING TRANS-SPLICING RIBOZYMES,” Haseloff et al., U.S. Pat. No. 5,863,774 “CELL ABLATION USING TRANS-SPLICING RIBOZYMES,” Haseloff et al., U.S. Pat. No. 5,849,548 “CELL ABLATION USING TRANS-SPLICING RIBOZYMES,” and Haseloff et al., U.S. Pat. No. 5,641,673 “CELL ABLATION USING TRANS-SPLICING RIBOZYMES.” Methods of ablating cells in vivo involving targeted trans-splicing to provide toxic products that generate sterile plants have also been described in, e.g., Haseloff et al., U.S. Pat. No. 5,866,384, supra. The techniques referenced above generally involve trans-splicing RNA sequences into native target RNAs.
Genetically male-sterile plants can be desirable for the production of hybrid seeds, because they avoid the need for expensive and laborious removal of, e.g., anthers from flowers to prevent self-fertilization. Transgenic methods of regenerating functionally male-sterile plants have included the development of pollen cells that are ablated specifically by the expression of fungal or bacterial ribonuclease transgenes fused to a pollen-specific promoter from the particular plant. Mariani, C. et al. (1992)
Nature
357, 384-387. See also, Haseloff et al., U.S. Pat. No. 5,866,384, supra.
In addition to trans-splicing RNAs, protein trans-splicing is also known. For example, certain modified proteins have been described which include “controllable intervening protein sequences” inserted into or adjacent to target proteins. Comb, et al. U.S. Pat. No. 5,834,247 “MODIFIED PROTEINS COMPRISING CONTROLLABLE INTERVENING PROTEIN SEQUENCES OR THEIR ELEMENTS METHODS OF PRODUCING SAME AND METHODS FOR PURIFICATION OF A TARGET PROTEIN COMPRISED BY A MODIFIED PROTEIN.” The inserted intervening sequences are capable of cleaving the modified protein in trans under controllable conditions, e.g., increased temperature, exposure to light, treatment with chemical reagents, etc. Furthermore, these intervening protein sequences can also be inserted into a target protein sequence so as to render the target inactive. Id. See also, Comb, et al. U.S. Pat. No. 5,496,714 “MODIFICATION OF PROTEIN BY USE OF A CONTROLLABLE INTERVENING PROTEIN SEQUENCE” and Belfort, U.S. Pat. No. 5,795,731 “INTEINS AS ANTIMICROBIAL TARGETS: GENETIC SCREENS FOR INTEIN FUNCTION.” Spontaneous (native) trans-splicing of both inteins and RNAs is also known.
More generally, relevant features of inteins and intein splicing, as well as certain forms of chemical ligation of polypeptides, are described in the abundant literature on the topics, including the references noted above and, e.g.: Clarke (1994) “A proposed mechanism for the self-splicing of proteins”
Proc. Natl. Acad. Sci. USA
91:11084-11088; Clyman (1995) “Some Microbes have splicing proteins”
ASM News
61:344-347; Colston and Davis (1994) “The ins and outs of protein splicing elements” Molecular Microbiology 12, 359-363; Cooper et al. (1993) “Protein splicing of the yeast TFP1 intervening protein sequence: a model for self-excision”
EMBO J.
12:2575-2583; Cooper and Stevens (1993) “Protein splicing: Excision of intervening sequences at the protein level”
BioEssays
15, 667-673; Cooper and Stevens (1995) “Protein splicing: Self-splicing of genetically mobile elements at the protein level”
TIBS
20, 351-357; Cook et al. (1995) “Photochemically initiated protein splicing”
Angew. Chem. Int. Ed. Engel
34, 1620-1630; Dalgaard, J. (1994) “Mobile introns and inteins: friend or foe?”
Trends Genet
10, 306-7; Davis et al. (1992) “Protein Splicing in the Maturation of M. Tuberculosis RecA Protein: A Mechanism for Tolerating a Novel Class of Intervening Sequence”
Cell
71:201-210; Davis et al. (1991) “Novel Structure of the recA Locus of
Mycobacterium tuberculosis
Implies Processing of the Gene Product”
J. Bacteriol.
173:5653-5662; Davis et al. (1994) “Evidence of selection for protein introns in the RecAs of pathogenic Mycobacteria”
EMBO J.
13, 699-703; Davis et al. (1995) “Protein splicing—the lengths some proteins will go to”
Antonie Van Leeuwenhoek
67:131-137; Doolittle, (1993) “The comings and goings of homing endonucleases and mobile introns”
Proc. Natl. Acad. Sci. USA.
90:5379-5381; Doolittle and Stoltzfus (1993) “Genes-in-pieces revisited”
Nature
361:403; Hirata and Anraku (1992) “Mutations at the Putative Junction Sites of the Yeast VMA1 Protein, the Catalytic Subunit of the Vacuolar Membrane H+−ATPase, Inhibit its Processing by Protein Splicing”
Biochem. Biophys. Res. Comm.
188:40-47; Hirata et al. (1990) “Molecular Structure of a Gene, VMA1, Encoding the Catalytic Subunit of H+−Translocating Adenosine Triphosphatase from Vacuolar Membranes of
Saccharomyces cereviaiae” J. Biol. Chem.
265, 6726-6733; Hodges et al. (1992) “Protein splicing removes intervening sequences in an archaea DNA polymerase”
Nucleic Acids Res.
20:6153-6157; Kane et al. (1990) “Protein Splicing Converts the Yeast TFP1 Gene Product to the 69-kD Subunit of the Vacuolar H+−Adenosine Triphosphatase”
Science
250:651-657; Koonin (1995) “A protein splice-junction motif in hedgehog family proteins”
Trends Biochem. Sci.
20:41-142; Kumar et al. (1996) “Functional characterization of the precursor and spliced forms of recA protein of
Mycobacterium tuberculosis” Biochemistry
35:1793-1802, and Kawasaki, M., et al., Biochemical and Biophysical Research Communications, vol. 222, “Folding-dependent in vitro protein splicing of the
Saccharomyces cerevisiae
VMA1 protozyme”, pp. 827-832, 1996. Gimble and Thorner (1992)
Nature
357:301-306; Gimble and Thorner (1993)
J. Biol. Chem.,
268:21844-2
Lassner Michael
Patten Phillip A.
Holman Christopher M.
Kruse Norman J.
Lu Frank
Maxyag, Inc.
Whisenant Ethan C.
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