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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

Scheme for the development of the pLL-EHC vector containing a centromeric DNA fragment for use as the backbone in AC constructs. (A) Structure of the pLL-EH vector. A 6,212-bp fragment between PciI and SalI was isolated from BAC vector pBeloBAC11. (B) Rice BAC clone 38J12 derived from the centromere of chromosome 8 [14]. (C) Ligation of FseI-digested pLL-EH and BAC 38J12 resulting in the vector pLL-EHC.
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Figure 1: Scheme for the development of the pLL-EHC vector containing a centromeric DNA fragment for use as the backbone in AC constructs. (A) Structure of the pLL-EH vector. A 6,212-bp fragment between PciI and SalI was isolated from BAC vector pBeloBAC11. (B) Rice BAC clone 38J12 derived from the centromere of chromosome 8 [14]. (C) Ligation of FseI-digested pLL-EH and BAC 38J12 resulting in the vector pLL-EHC.

Mentions: We first developed the pLL-EH vector (Figure 1A). This vector (12,012 bp) consists of two DNA fragments. The first fragment (6,212-bp) was isolated from the BAC pBeloBAC11 [13] by double digestion with PciI and SalI (Figure 1A). This fragment contains all the genes required for stable propagation and maintenance of large DNA fragments in E. coli. The second fragment was synthesized containing a number of restriction sites for cloning and recombination. A hygromycin resistance gene (Hpt) and a reporter gene Egfp were also inserted into this fragment (Figure 1A). The attP1 site can be used for in vitro site-specific recombination with the attB1 site from telomeric DNA vectors. The lox71 and the φC31 attB1 sites can be used to insert additional DNA sequences into the vector or future potential artificial chromosomes of transgenic plants.


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)

Scheme for the development of the pLL-EHC vector containing a centromeric DNA fragment for use as the backbone in AC constructs. (A) Structure of the pLL-EH vector. A 6,212-bp fragment between PciI and SalI was isolated from BAC vector pBeloBAC11. (B) Rice BAC clone 38J12 derived from the centromere of chromosome 8 [14]. (C) Ligation of FseI-digested pLL-EH and BAC 38J12 resulting in the vector pLL-EHC.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3101654&req=5

Figure 1: Scheme for the development of the pLL-EHC vector containing a centromeric DNA fragment for use as the backbone in AC constructs. (A) Structure of the pLL-EH vector. A 6,212-bp fragment between PciI and SalI was isolated from BAC vector pBeloBAC11. (B) Rice BAC clone 38J12 derived from the centromere of chromosome 8 [14]. (C) Ligation of FseI-digested pLL-EH and BAC 38J12 resulting in the vector pLL-EHC.
Mentions: We first developed the pLL-EH vector (Figure 1A). This vector (12,012 bp) consists of two DNA fragments. The first fragment (6,212-bp) was isolated from the BAC pBeloBAC11 [13] by double digestion with PciI and SalI (Figure 1A). This fragment contains all the genes required for stable propagation and maintenance of large DNA fragments in E. coli. The second fragment was synthesized containing a number of restriction sites for cloning and recombination. A hygromycin resistance gene (Hpt) and a reporter gene Egfp were also inserted into this fragment (Figure 1A). The attP1 site can be used for in vitro site-specific recombination with the attB1 site from telomeric DNA vectors. The lox71 and the φC31 attB1 sites can be used to insert additional DNA sequences into the vector or future potential artificial chromosomes of transgenic plants.

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