Progress 10/01/00 to 09/30/04
Outputs
Telomeres and centromeres are the two most important landmarks of eukaryotic chromosomes. Telomeres serve as inert caps, preventing chromosomal rearrangements stemming from the reactive and recombinogenic behavior of broken chromosome ends. Centromeres are responsible for chromosome movement in both mitosis and meiosis by serving as an attachment site for spindle fibers. Thus, telomeres and centromeres play key roles for the proper transmission and segregation of chromosomes in mitosis and meiosis, thereby ensuring equal distribution of genetic information to the next generation. Cloning of functional telomeres and centromeres is also an essential step to construct artificial chromosomes, which have attracted tremendous interests from both academia and the biotech industry to develop plant artificial chromosomes (PACs). The main goal of this project was to characterize the telomeric and centromeric DNA in potato and to develop a foundation for construction of PACs
using potato as a model. We successfully developed a special strategy to isolate centromeric DNA repeats in potato. Our approach was to search the most dominant tandem repeats in the potato genome using a bacterial artificial chromosome (BAC) library-based screening method. A total of 25 putative potato centromeric BAC clones were isolated and analyzed by fluorescence in situ hybridization (FISH) to visualize their chromosomal locations. Nineteen of these clones showed strong centromeric and pericentromeric signals. Plasmid libraries have been developed from several centromeric BAC clones and a number of centromeric repeats have been isolated from these libraries. We have also isolated a telomere-similar DNA element, pSbTC1, located in the centromere of potato chromosomes. Most notably, the pSbTC1-related sequences have undergone extensive amplification and a single array can span up to several megabases. These results suggest that the pSbTC1-related sequences are not simple relics of
ancient events in karyotype evolution, such as chromosome fusions. It will be interesting to investigate if the pSbTC1-related sequences are involved in centromere function. We have also investigated the methods to deliver large DNA fragments into potato cells. Currently only the binary bacterial artificial chromosome (BIBAC) and transformation-competent artificial chromosome (TAC) vectors can be used in Agrobacterium-mediated transformation with large DNA fragments. To test the BIBAC and TAC systems in potato transformation we selected six potato BAC clones with insert sizes ranging from 110 kb to 180 kb for BIBAC and TAC construct development. We found that all of the BIBAC and TAC clones can be stably maintained in E. coli. However, when these BIBAC and TAC clones were transformed into A. tumefaciens strains, the inserts of the clones were all significantly deleted. We determined that the insert size was the major factor that impacts their stability in A. tumefaciens. We also
developed a transposon-based technique that can be used to efficiently subclone a BAC insert into two to three BIBAC/TAC constructs to circumvent the instability problem.
Impacts
Centromeres govern the proper segregation and transmission of chromosomes during cell division. Cloning and characterization of plant centromeric DNA is essential to understand the structure and organization of plant chromosomes and is a critical step to develop plant artificial chromosomes (PACs). Potato is the optimal plant model for PAC construction because it is asexually propagated, tolerant to aneuploidy, and is highly transformable. The isolation of potato BAC clones containing centromeric DNA is the first step toward our ultimate goal to develop a PAC in potato. Our technique for centromeric DNA isolation can potentially be applied in other eukaryotic species. Successful applications of PACs will rely on techniques to deliver large DNA fragments into plant cells. Our work has exploited the limit and potential of the current BIBAC and TAC systems. We also demonstrated that the currently available Agrobacterium strains are not ideal to hold large insert plasmids.
We need to continue to engineer these stains to increase the stability of large insert constructs.
Publications
- Stupar, R.M., Song, J., Tek, A.L., Cheng, Z.K., Dong, F., and Jiang, J. (2002) Highly condensed potato pericentromeric heterochromatin contains rDNA-related tandem repeats. Genetics 162: 1435-1444.
- Song, J., Bradeen, J.M., Naess, S.K., Helgeson, J.P., and Jiang, J. (2003) BIBAC and TAC clones containing potato genomic DNA fragments larger than 100 kb are not stable in Agrobacterium. Theor. Appl. Genet. 107: 958-964.
- Tek, A.L., Stevenson, W.R., Helgeson, J.P., and Jiang, J. (2004) Introgression of tuber soft rot and early blight resistances from Solanum brevidens into cultivated potato. Theor. Appl. Genet. 109: 249-254.
- Tek, A.L. and Jiang, J. (2004) The centromeric regions of potato chromosomes contain megabase-sized tandem arrays of telomere-similar sequence. Chromosoma 113: 77-83.
- Dong, F., Tek, A.L., Frasca, A.B.L., McGrath, J.M., Wielgus, S.M., Helgeson, J.P., and J. Jiang (2005) Development and characterization of potato-Solanum brevidens chromosomal addition/substitution lines. Cytogenet. Genome Res. (in press)
- Tek, A.L., Song, J., Macas, J. and Jiang, J. (2005) Sobo, a recently amplified satellite repeat and its implications on origin of tandemly repeated sequences. Genetics (submitted).
- Song, J., Dong, F., Lilly, J.W., Stupar, R.M., and Jiang, J. (2001) Instability of bacterial artificial chromosome (BAC) clones containing tandemly repeated DNA sequences. Genome 44: 463-469.
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Progress 01/01/03 to 12/31/03
Outputs
Efficient methods to deliver large DNA fragments into plant cells will be crucial for future plant artificial chromosome projects. Currently only the binary bacterial artificial chromosome (BIBAC) and transformation-competent artificial chromosome (TAC) vectors can be used in Agrobacterium-mediated transformation with large DNA fragments. To test the BIBAC and TAC systems in potato transformation we selected six potato BAC clones, including two clones derived from centromeric regions. The inserts of these six clones, ranging from 110-180 kb, were transferred into BIBAC and TAC vectors. We found that all of the BIBAC and TAC clones can be stably maintained in E. coli. However, when these BIBAC and TAC clones were transformed into A. tumefaciens strains, the inserts of the clones were all significantly deleted. We examined several possible factors that may cause this instability, including the insert sizes of the BIBAC and TAC constructs, potato DNA fragments consisting
of highly repetitive or largely single copy DNA sequences, different Agrobacterium transformation methods, and different Agrobacterium strains. The insert sizes of the potato BIBAC and TAC constructs were found to be critical to their stability in Agrobacterium. All constructs containing a potato DNA fragment larger than 100 kb were not stable in any of the four tested Agrobacterium strains, including two recA-deficient strains. In a previous report a 150-kb human BIBAC2 clone can be stably maintained in Agrobacterium strains. Thus, the relationship between the sizes of BIBAC/TAC constructs and their stability in Agrobacterium may vary in different plant species. We developed a transposon-based technique that can be used to efficiently subclone a BAC insert into two to three BIBAC/TAC constructs to circumvent the instability problem. In our potato BAC library, more than 99% of the clones lack internal NotI sites within the BAC inserts. Our transposon-based approach can effectively
introduce one or more NotI restriction sites into BAC inserts for subsequent size manipulation. This technique should be equally applicable to other plasmids, such as P1 clones and P1-derived artificial chromosome clones. We continued our effort to isolate DNA elements located in the centromeric regions of potato chromosomes. We have isolated an interstitial telomeric repeat (ITR) element from a diploid potato species Solanum bulbocastanum. This ITR element is located in the centromeric regions and is widely distributed in Solanum species. We quantified this ITR element in three different Solanum species and demonstrated that a single tandem array of this ITR can span up to several megabases. Detailed molecular and cytogenetic characterization of this element is underway.
Impacts
Plant artificial chromosomes (PACs) will be a ground-breaking tool to study structure and function of plant chromosomes. PACs will also have a major impact on plant genetic engineering in the future. Successful applications of PACs will rely on techniques to deliver large DNA fragments into plant cells. Our work has exploited the limit and potential of the current BIBAC and TAC systems. We also demonstrated that the currently available Agrobacterium strains are not ideal to hold large insert plasmids. We need to continue to engineer these strains to increase the stability of large insert constructs.
Publications
- Song, J., Bradeen, J.M., Naess, S.K., Helgeson, J.P., and Jiang, J. (2003) BIBAC and TAC clones containing potato genomic DNA fragments larger than 100 kb are not stable in Agrobacterium. Theor. Appl. Genet. 107: 958-964.
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Progress 01/01/02 to 12/31/02
Outputs
We continued our effort to isolate DNA elements located in the centromeric and pericentromeric regions of potato chromosomes. We have fully characterized the 2D8 element located in the pericentromeric regions of several potato chromosomes. It was very surprising that the 5.9-kb 2D8 repeat is highly homologous to the intergenic spacer (IGS) of the 18S.25S ribosomal RNA genes in potato. This repeat is associated with highly condensed pericentromeric heterochromatin at several hemizygous loci. The 2D8 repeat is highly variable in structure and copy number throughout the Solanum genus, suggesting that it is evolutionarily dynamic. We identified two additional repeats, 4A4 and 26J19, which are also related to potato rDNA. The 4A4 and 26J19 repeats are located at the same chromosomal regions as the 2D8 repeat but do not cross hybridize to the 2D8 loci. Our results showed that rDNA-related repetitive DNA elements are widely spread in the potato genome. We have developed a
special strategy to isolate centromeric DNA repeats in potato. Our approach is to search the most dominant tandem repeats in the potato genome using a bacterial artificial chromosome (BAC) library-based screening method. We tested this screening method in both Arabidopsis thaliana and rice. Centromere-related satellite DNA was successfully recovered in both species. A total of 25 putative centromeric BAC clones were analyzed by fluorescence in situ hybridization (FISH) to visualize their chromosomal locations. To our surprise 19 clones showed strong centromeric and pericentromeric signals. These 19 BACs can be divided into four groups based on the FISH patterns. Molecular and cytogenetic characterization of these BAC clones are underway.
Impacts
Heterochromatin accounts for a significant part of higher eukaryotic genomes, yet the origin and evolution of the DNA sequences in the heterochromatic regions is poorly understood. We discovered a unique class of pericentromeric heterochromatin consisting of sequences related to the intergenic spacer of the 18S.25S ribosomal RNA genes in potato. This is the first report on a cytologically defined plant heterochromatin feature in which the origin of its DNA sequences is revealed. These results have important implications on satellite DNA and heterochromatin formation in the plant genomes. Centromeres govern the proper segregation and transmission of chromosomes during cell division. Cloning and characterization of plant centromeric DNA is essential to understand the structure and organization of plant chromosomes and is a critical step to develop plant artificial chromosomes (PACs). Potato is the optimal plant model for PAC construction because it is asexually
propagated, tolerant to aneuploidy, and is highly transformable. The isolation of potato BAC clones containing centromeric DNA is the first step toward our ultimate goal to develop a PAC in potato. Our technique for centromeric DNA isolation can potentially be applied in other eukaryotic species.
Publications
- Stupar, R.M., Song, J., Tek, A.L., Cheng, Z.K., Dong, F., and Jiang, J. (2002) Highly condensed potato pericentromeric heterochromatin contains rDNA-related tandem repeats. Genetics 162: 1435-1444.
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Progress 01/01/01 to 12/31/01
Outputs
In order to identify potato bacterial artificial chromosome (BAC) clones containing highly repetitive sequences, including those associated with centromeric and subtelomeric regions, filters prepared from a S. bulbocastanum BAC library were screened using a small amount of potato genomic DNA as a probe. Clone 2D8 showed one of the strongest hybridization signals to the genomic DNA, suggesting that this BAC may contain one of the most repetitive DNA elements in the potato genome. Pulse-field gel electrophoresis analysis showed that the insert size of 2D8 is approximately 230 kb. However, the HindIII restriction pattern of 2D8 gave only three bands: a strong band at 5.9 kb, a relatively weaker band at 8.8 kb, and the BAC vector band at 7.4 kb. These results indicate that the insert of BAC 2D8 contains mainly a 5.9-kb tandem repeat. This 5.9-kb band was subcloned for further analysis and was named the "2D8 repeat". Fluorescence in situ hybridization analysis revealed
that the 2D8 repeat is associated with highly condensed and knob-like pericentromeric heterochromatin. One 5.9-kb 2D8 subclone was completely sequenced. This 5,862-bp sequence consists of two degenerate monomers of similar size and composition. BLAST search revealed a high sequence homology (E-value=2e-26) of the two monomers within the 2D8 repeat to the intergenic spacer (IGS) sequence of the 18S.25S ribosomal RNA genes of potato. Significant expansion of DNA sequences, which were previously identified as DNA amplification-promoting elements within tobacco and potato rDNA IGS, was discovered in the 2D8 repeat. The unique structural features associated with the 2D8 repeat suggest that the 2D8 loci may originate through DNA amplification mechanisms. Gel-blot hybridization analysis revealed copy number and structure polymorphisms of the 2D8 repeat across wild and cultivated Solanum species, indicating that this repeat is evolutionarily dynamic.
Impacts
Heterochromatin accounts for a significant part of higher eukaryotic genomes, yet the origin and evolution of the DNA sequences in the heterochromatic regions is poorly understood. We discovered a unique class of pericentromeric heterochromatin consisting of orphon DNA derived from the intergenic spacer of the 18S.25S ribosomal RNA genes in potato. This is the first report on a cytologically defined plant heterochromatin feature in which the origin of its DNA sequences is revealed. This discovery will shed lights on the origin and evolution of heterochromatic DNA in higher eukaryotic genomes.
Publications
- Dong, F., McGrath, J.M., Helgeson, J.P., and Jiang, J. (2001) The genetic identity of alien chromosomes in potato breeding lines revealed by sequential GISH and FISH analyses using chromosome-specific cytogenetic DNA markers. Genome 44: 729-734.
- Song, J., Dong, F., Lilly, J.W., Stupar, R.M., and Jiang, J. (2001) Instability of bacterial artificial chromosome (BAC) clones containing tandemly repeated DNA sequences. Genome 44: 463-469.
- Fu, H.Y., Du, J., Song, J.Q., Jiang, J., and Park, W.D. (2001) Potato and tomato Forever Young genes contain class-I patatin promoter-like sequences. Botanical Bulletin of Academia Sinica 42: 231-241.
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