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WO 2011/011693 Al (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date 27 January 2011 (27.01.2011) WO 2011/011693 Al (51) International Patent Classification: (74) Agent: ZINKL, Gregory; Chromatin, Inc., 3440 South C12Q 1/68 (2006.01) AOlH 5/00 (2006.01) Dearborn Street, Suite 010, Chicago, Illinois 60616 (US). (21) International Application Number: (81) Designated States (unless otherwise indicated, for every PCT/US20 10/043065 kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, (22) Date: International Filing CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, 23 July 2010 (23.07.2010) DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, (25) Filing Language: English HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, (26) Publication Language: English ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, (30) Priority Data: NO, NZ, OM, PE, PG, PH, PL, PT, RO, RS, RU, SC, SD, 61/228,015 23 July 2009 (23.07.2009) US SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (71) Applicant (for all designated States except US): CHRO¬ MATIN, INC. [US/US]; 3440 South Dearborn Street, (84) Designated States (unless otherwise indicated, for every Suite 010, Chicago, IL 60616 (US). kind of regional protection available): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, (72) Inventors; and ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, (75) Inventors/Applicants (for US only): PREUSS, Daphne TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, [US/US]; 520 W. Huron # 119, Chicago, Illinois 60654 EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, (US). BARONE, Pierluigi [IT/US]; 724 Clifton Dr., LV, MC, MK, MT, NL, NO, PL, PT, RO, SE, SI, SK, Charleston, Illinois 61290 (US). CARLSON, Shawn SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, [US/US]; PO Box 121, 307. 5 W. Chestnut St., Bondville, GW, ML, MR, NE, SN, TD, TG). Illinois 61815 (US). COPENHAVER, Gregory, P. [US/ US]; 23 15 Mountside Dr., Chapel Hill, North Carolina Published: 2751 6 (US). LUO, Song [CN/US]; 917 E. 54th PL, — with international search report (Art. 21(3)) Chicago, Illinois 60615 (US). MACH, Jennifer — with sequence listing part of description (Rule 5.2(a)) [US/US]; 2958 N. Kilbourn Ave, Chicago, Illinois 60641 (US). (54) Title: SORGHUM CENTROMERE SEQUENCES AND MINICHROMOSOMES (57) Abstract: The invention is generally related to MCs containing sorghum centromere sequences. In addition, the invention provides for methods of generating plants transformed with these MCs. MCs with novel compositions and structures are used to transform plants cells which are in turn used to generate the plant. Methods for generating the plant include methods for deliver - ing the MC into plant cell to transform the cell, methods for selecting the transformed cell, and methods for isolating plants trans formed with the MC. SORGHUM CENTROMERE SEQUENCES AND MINICHROMOSOMES CROSS-REFERENCE TO RELATED APPLICATIONS [001] This application claims priority t o US Provisional Patent Application by D. Preuss et ai, US Patent Application Serial No. 61/228,015, titled, "SORGHUM CENTROMERE SEQUENCES AND MINICHROMOSOMES," filed July 23, 2009, which is incorporated by reference herein in its entirety. GOVERNMENT SUPPORT [002] Not applicable. COMPACT DISC FOR SEQUENCE LISTINGS AND TABLES [003] Not applicable. FIELD O F THE INVENTION [004] The present invention relates t o sorghum centromere sequences that are useful, for example, in constructing artificial chromosomes comprising sorghum centromere sequences, and cells and organisms comprising such artificial chromosomes, including Sorghum bicolor and Sorghum Sudanese. Methods the make and use the disclosed sorghum centromeres are also disclosed. BACKGROUND OFTHE INVENTION [005] Two general approaches are used for introduction of new heritable genetic information ("transformation") into cells. One approach is t o introduce the new genetic information as part of another DNA molecule, referred t o as an "episomal vector," or "minichromosome" (MC), which can be maintained as an independent unit (an episome) apart from the host chromosomal DNA molecule(s). Episomal vectors contain all the necessary DNA sequence elements required for DNA replication and maintenance of the vector within the cell. Many episomal vectors are available for use in bacterial cells (for example, see Maniatis et ai, Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982). However, only a few episomal vectors that function in higher eukaryotic cells have been developed. Higher eukaryotic episomal vectors were primarily based on naturally occurring viruses. In higher plant systems gemini viruses are double-stranded DNA viruses that replicate through a double-stranded intermediate upon which an episomal vector could be based, although the gemini virus is limited t o an approximately 800 bp insert. Although an episomal plant vector based on the Cauliflower Mosaic Virus has been developed, its capacity t o carry new genetic information also is limited (Brisson etai, Nature, 310:511, 1984). [006] The other general method of genetic transformation involves integration of introduced DNA sequences into the recipient cell's chromosomes, permitting the new information t o be replicated and partitioned to the cell's progeny as a part of the natural chromosomes. The introduced DNA usually can be broken and joined together in various combinations before it is integrated at random sites into the cell's chromosome (see, for example Wigler et al., Cell, 11:223, 1977). Common problems with this procedure are the rearrangement of introduced DNA sequences and unpredictable levels of expression due t o the location of the transgene integration site in the host genome or so called "position effect variegation" (Shingo etai, MoI. Cell. Biol., 6:1787, 1986). Further, unlike episomal DNA, integrated DNA cannot normally be precisely removed. A more refined form of integrative transformation can be achieved by exploiting naturally occurring viruses that integrate into the host's chromosomes as part of their life cycle, such as retroviruses (see Chepko etal., Cell, 37:1053, 1984). [007] One common genetic transformation method used in higher plants is based on the transfer of bacterial DNA into plant chromosomes that occurs during infection by the phytopathogenic soil bacterium Agrobacterium (see Nester etal., Ann. Rev. Plant Phys., 35:387-413, 1984). By substituting genes of interest for a portion of the naturally transferred bacterial sequences (called T-DNA), investigators have been able to introduce new DNA into plant cells. However, even this more "refined" integrative transformation system is limited in three major ways. First, DNA sequences introduced into plant cells using the Agrobacterium T-DNA system are frequently rearranged (see Jones et al., MoI Gen. Genet., 207:478, 1987). Second, the expression of the introduced DNA sequences varies between individual transformants (see Jones et al., EMBOJ., 4:2411-2418, 1985). This variability is presumably caused by rearranged sequences and the influence of surrounding sequences in the plant chromosome (i.e., position effects), as well as methylation of the transgene. Finally, insertion of extra elements into the genome can disrupt the genes, promoters or other genetic elements necessary for normal plant growth and function. [008] Another widely used technique to genetically transform plants involves the use of microprojectile bombardment to integrate DNA sequences into the genome. In this process, a nucleic acid containing the desired genetic elements to be introduced into the plant's native chromosome is deposited on or in small metallic particles, e.g., tungsten, platinum, or preferably gold, which are then delivered at a high velocity into the plant tissue or plant cells. However, similar problems arise as with Agrobacterium-me άlate ά gene transfer, and as noted above expression of the inserted DNA can be unpredictable and insertion of extra elements into the genome can disrupt and adversely impact plant processes. [009] One attractive alternative to the commonly used methods of transformation is the use of an artificial chromosome. Artificial chromosomes are episomal nucleic acid molecules that exist autonomously from the native chromosomes of the host genome. They can be linear or circular DNA molecules that are comprised of cis-acting nucleic acid sequence elements that provide replication and partitioning activities (see Murray etal., Nature, 305:189-193, 1983). Desired elements include: (1) origin of replication, which are the sites for initiation of DNA replication, (2) centromeres (site of kinetochore assembly and responsible for proper distribution of replicated chromosomes into daughter cells at mitosis or meiosis), and (3) if the chromosome is linear, telomeres (specialized DNA structures at the ends of linear chromosomes that function t o stabilize the ends and facilitate the complete replication of the extreme termini of the DNA molecule). An additional desired element is a chromatin organizing sequence. It is well documented that centromere function is crucial for stable chromosomal inheritance in almost all eukaryotic organisms (reviewed in Nicklas, J Cell Sci. 189:283-5, 1988). The centromere accomplishes this by attaching, via centromere binding proteins, to the spindle fibers during mitosis and meiosis, thus ensuring proper gene segregation during cell divisions. [010] Artificial chromosomes have been engineered using one of two approaches. The first approach identifies and assembles the desired chromosomal elements into an artificial construct. This is approach has been described as "bottom-up" and involves the use of a heterologous system (i.e. bacteria or fungal) to perform the various cloning steps necessary to assemble the artificial chromosome.
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