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De novo assembly of potential linear artificial chromosome constructs capped with expansive telomeric repeats.

Lin L, Koo DH, Zhang W, St Peter J, Jiang J - Plant Methods (2011)

Bottom Line: The centromeric DNA backbone can be ligated with the telomeric DNA fragments to generate AC constructs consisting of a large centromeric DNA fragment capped with expansive telomeric DNA at both ends.We successfully developed a procedure that circumvents the problem of cloning and maintaining long arrays of telomeric DNA sequences that are not stable in E. coli.Our procedure allows development of AC constructs in different eukaryotic species that are capped with long and designed sizes of telomeric DNA fragments.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Horticulture, University of Wisconsin-Madison, Madison, WI 53706, USA. jjiang1@wisc.edu.

ABSTRACT

Background: Artificial chromosomes (ACs) are a promising next-generation vector for genetic engineering. The most common methods for developing AC constructs are to clone and combine centromeric DNA and telomeric DNA fragments into a single large DNA construct. The AC constructs developed from such methods will contain very short telomeric DNA fragments because telomeric repeats can not be stably maintained in Escherichia coli.

Results: We report a novel approach to assemble AC constructs that are capped with long telomeric DNA. We designed a plasmid vector that can be combined with a bacterial artificial chromosome (BAC) clone containing centromeric DNA sequences from a target plant species. The recombined clone can be used as the centromeric DNA backbone of the AC constructs. We also developed two plasmid vectors containing short arrays of plant telomeric DNA. These vectors can be used to generate expanded arrays of telomeric DNA up to several kilobases. The centromeric DNA backbone can be ligated with the telomeric DNA fragments to generate AC constructs consisting of a large centromeric DNA fragment capped with expansive telomeric DNA at both ends.

Conclusions: We successfully developed a procedure that circumvents the problem of cloning and maintaining long arrays of telomeric DNA sequences that are not stable in E. coli. Our procedure allows development of AC constructs in different eukaryotic species that are capped with long and designed sizes of telomeric DNA fragments.

No MeSH data available.


Related in: MedlinePlus

DNA composition of the AC constructs. (A) PFGE of AC constructs. (B) Southern blot hybridized with a telomeric DNA probe. (C) Southern blot hybridized with the rice centromeric satellite repeat CentO. Lane 1: Linear ACs capped with ~2 to 5-kb telomeric DNA at both ends. Lane 2: Mixture of pLL-EHC and back-to-back telomeric DNA without recombination. Note: the large bands associated with pLL-EHC did not hybridize to the telomeric DNA probe in (B). Lane 3: Linear molecules derived from recombination between pLL-EHC and linearized pLL-BKE (~10-kb of non-telomeric DNA, see Experimental Procedures). The arrowhead in (A) points to the non-recombined pLL-BKE molecules. Lane 4: pLL-EHC DNA after incubation with BP clonase. Lane 5: Linear AC constructs capped with ~5 to 10-kb telomeric DNA at the left arm only after recombination between pLL-EHC and expansive telomeric DNA from pLL-TBS. The red square in (B) includes the non-recombined telomeric DNA molecules. Lane 6: Linear AC constructs capped with ~5 to 10-kb telomeric DNA at the right arm only after recombination between pLL-EHC and expansive telomeric DNA from pLL-TSB. The red square in (B) includes the non-recombined telomeric DNA molecules.
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Figure 5: DNA composition of the AC constructs. (A) PFGE of AC constructs. (B) Southern blot hybridized with a telomeric DNA probe. (C) Southern blot hybridized with the rice centromeric satellite repeat CentO. Lane 1: Linear ACs capped with ~2 to 5-kb telomeric DNA at both ends. Lane 2: Mixture of pLL-EHC and back-to-back telomeric DNA without recombination. Note: the large bands associated with pLL-EHC did not hybridize to the telomeric DNA probe in (B). Lane 3: Linear molecules derived from recombination between pLL-EHC and linearized pLL-BKE (~10-kb of non-telomeric DNA, see Experimental Procedures). The arrowhead in (A) points to the non-recombined pLL-BKE molecules. Lane 4: pLL-EHC DNA after incubation with BP clonase. Lane 5: Linear AC constructs capped with ~5 to 10-kb telomeric DNA at the left arm only after recombination between pLL-EHC and expansive telomeric DNA from pLL-TBS. The red square in (B) includes the non-recombined telomeric DNA molecules. Lane 6: Linear AC constructs capped with ~5 to 10-kb telomeric DNA at the right arm only after recombination between pLL-EHC and expansive telomeric DNA from pLL-TSB. The red square in (B) includes the non-recombined telomeric DNA molecules.

Mentions: To confirm the recombination between the back-to-back telomeric DNA fragment and the pLL-EHC plasmid specific PCR primers were designed from the junction regions based on the backbone sequences of plasmids pLL-EHC, pLL-TBS, and pLL-TSB (Figure 4). The linear AC constructs were isolated by pulsed field gel electrophoresis (PFGE). PCR amplification using the junction-specific primers resulted in DNA fragments matching expected sizes (Figure 4). The amplified PCR fragments were also confirmed by sequencing analysis (data not shown). We also developed linear constructs consisting of centromeric DNA capped with telomeric DNA at only one of the two ends. The single junction associated these constructs was also confirmed by PCR analysis (Figure 4). Southern blot hybridization analysis showed that only AC constructs with telomeric DNA attached at one or both ends hybridized to both telomeric and centromeric DNA probes (Figure 5).


De novo assembly of potential linear artificial chromosome constructs capped with expansive telomeric repeats.

Lin L, Koo DH, Zhang W, St Peter J, Jiang J - Plant Methods (2011)

DNA composition of the AC constructs. (A) PFGE of AC constructs. (B) Southern blot hybridized with a telomeric DNA probe. (C) Southern blot hybridized with the rice centromeric satellite repeat CentO. Lane 1: Linear ACs capped with ~2 to 5-kb telomeric DNA at both ends. Lane 2: Mixture of pLL-EHC and back-to-back telomeric DNA without recombination. Note: the large bands associated with pLL-EHC did not hybridize to the telomeric DNA probe in (B). Lane 3: Linear molecules derived from recombination between pLL-EHC and linearized pLL-BKE (~10-kb of non-telomeric DNA, see Experimental Procedures). The arrowhead in (A) points to the non-recombined pLL-BKE molecules. Lane 4: pLL-EHC DNA after incubation with BP clonase. Lane 5: Linear AC constructs capped with ~5 to 10-kb telomeric DNA at the left arm only after recombination between pLL-EHC and expansive telomeric DNA from pLL-TBS. The red square in (B) includes the non-recombined telomeric DNA molecules. Lane 6: Linear AC constructs capped with ~5 to 10-kb telomeric DNA at the right arm only after recombination between pLL-EHC and expansive telomeric DNA from pLL-TSB. The red square in (B) includes the non-recombined telomeric DNA molecules.
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Related In: Results  -  Collection

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Figure 5: DNA composition of the AC constructs. (A) PFGE of AC constructs. (B) Southern blot hybridized with a telomeric DNA probe. (C) Southern blot hybridized with the rice centromeric satellite repeat CentO. Lane 1: Linear ACs capped with ~2 to 5-kb telomeric DNA at both ends. Lane 2: Mixture of pLL-EHC and back-to-back telomeric DNA without recombination. Note: the large bands associated with pLL-EHC did not hybridize to the telomeric DNA probe in (B). Lane 3: Linear molecules derived from recombination between pLL-EHC and linearized pLL-BKE (~10-kb of non-telomeric DNA, see Experimental Procedures). The arrowhead in (A) points to the non-recombined pLL-BKE molecules. Lane 4: pLL-EHC DNA after incubation with BP clonase. Lane 5: Linear AC constructs capped with ~5 to 10-kb telomeric DNA at the left arm only after recombination between pLL-EHC and expansive telomeric DNA from pLL-TBS. The red square in (B) includes the non-recombined telomeric DNA molecules. Lane 6: Linear AC constructs capped with ~5 to 10-kb telomeric DNA at the right arm only after recombination between pLL-EHC and expansive telomeric DNA from pLL-TSB. The red square in (B) includes the non-recombined telomeric DNA molecules.
Mentions: To confirm the recombination between the back-to-back telomeric DNA fragment and the pLL-EHC plasmid specific PCR primers were designed from the junction regions based on the backbone sequences of plasmids pLL-EHC, pLL-TBS, and pLL-TSB (Figure 4). The linear AC constructs were isolated by pulsed field gel electrophoresis (PFGE). PCR amplification using the junction-specific primers resulted in DNA fragments matching expected sizes (Figure 4). The amplified PCR fragments were also confirmed by sequencing analysis (data not shown). We also developed linear constructs consisting of centromeric DNA capped with telomeric DNA at only one of the two ends. The single junction associated these constructs was also confirmed by PCR analysis (Figure 4). Southern blot hybridization analysis showed that only AC constructs with telomeric DNA attached at one or both ends hybridized to both telomeric and centromeric DNA probes (Figure 5).

Bottom Line: The centromeric DNA backbone can be ligated with the telomeric DNA fragments to generate AC constructs consisting of a large centromeric DNA fragment capped with expansive telomeric DNA at both ends.We successfully developed a procedure that circumvents the problem of cloning and maintaining long arrays of telomeric DNA sequences that are not stable in E. coli.Our procedure allows development of AC constructs in different eukaryotic species that are capped with long and designed sizes of telomeric DNA fragments.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Horticulture, University of Wisconsin-Madison, Madison, WI 53706, USA. jjiang1@wisc.edu.

ABSTRACT

Background: Artificial chromosomes (ACs) are a promising next-generation vector for genetic engineering. The most common methods for developing AC constructs are to clone and combine centromeric DNA and telomeric DNA fragments into a single large DNA construct. The AC constructs developed from such methods will contain very short telomeric DNA fragments because telomeric repeats can not be stably maintained in Escherichia coli.

Results: We report a novel approach to assemble AC constructs that are capped with long telomeric DNA. We designed a plasmid vector that can be combined with a bacterial artificial chromosome (BAC) clone containing centromeric DNA sequences from a target plant species. The recombined clone can be used as the centromeric DNA backbone of the AC constructs. We also developed two plasmid vectors containing short arrays of plant telomeric DNA. These vectors can be used to generate expanded arrays of telomeric DNA up to several kilobases. The centromeric DNA backbone can be ligated with the telomeric DNA fragments to generate AC constructs consisting of a large centromeric DNA fragment capped with expansive telomeric DNA at both ends.

Conclusions: We successfully developed a procedure that circumvents the problem of cloning and maintaining long arrays of telomeric DNA sequences that are not stable in E. coli. Our procedure allows development of AC constructs in different eukaryotic species that are capped with long and designed sizes of telomeric DNA fragments.

No MeSH data available.


Related in: MedlinePlus