Highly selective isolation of human DNAs from rodent–human hybrid cells as circular yeast artificial chromosomes by transformation-associated recombination cloning

Larionov, Vladimir; Kouprina, Natalya; Graves, Joan P.; Resnick, Michael A.

doi:10.1073/pnas.93.24.13925

Cited by 50 publications

(69 citation statements)

References 24 publications

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“…Southern blot hybridization with a probe of labeled total human DNA showed that the circular YACs derived from YAC77 ranged from ∼50 to 700 kb. This is similar to the size range of YACs (60 to 700 kb) that were recovered previously with the pNKBAC39 vector during TAR cloning of human DNA from hybrid cell lines containing whole human chromosomes (9,24). Similar results were obtained by linearizing the YACs by NotI digestion with an additional set of clones.…”

Section: Overlapping Circular Bacs From a Megabase Size Linear Yac Gesupporting

confidence: 72%

“…Frequency of loss of this chromosome was no higher than once per 1000 cell divisions. Circular YACs with human and mouse DNA inserts were created either by in vivo ligation (26) or recombination in yeast (9,24,27). While there are no systematic studies, it is clear from published data that circular molecules up to ∼1.0 Mb can be constructed in yeast, and the mitotic stability of these circles is no different from linear YACs of the same size.…”

Section: Discussionmentioning

confidence: 99%

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Recovery and potential utility of YACs as circular YACs/BACs

Cocchia¹

2000

Nucleic Acids Research

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A method has been established to convert pYAC4-based linear yeast artificial chromosomes (YACs) into circular chromosomes that can also be propagated in Escherichia coli cells as bacterial artificial chromosomes (BACs). The circularization is based on use of a vector that contains a yeast dominant selectable marker (G418R), a BAC cassette and short targeting sequences adjacent to the edges of the insert in the pYAC4 vector. When it is introduced into yeast, the vector recombines with the YAC target sequences to form a circular molecule, retaining the insert but discarding most of the sequences of the YAC telomeric arms. YACs up to 670 kb can be efficiently circularized using this vector. Re-isolation of megabase-size YAC inserts as a set of overlapping circular YAC/BACs, based on the use of an Alu-containing targeting vector, is also described. We have shown that circular DNA molecules up to 250 kb can be efficiently and accurately transferred into E.coli cells by electroporation. Larger circular DNAs cannot be moved into bacterial cells, but can be purified away from linear yeast chromosomes. We propose that the described system for generation of circular YAC derivatives can facilitate sequencing as well as functional analysis of genomic regions.

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Section: Overlapping Circular Bacs From a Megabase Size Linear Yac Gesupporting

confidence: 72%

Section: Discussionmentioning

confidence: 99%

Recovery and potential utility of YACs as circular YACs/BACs

Cocchia¹

2000

Nucleic Acids Research

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“…Similar requirement for homology is seen in gene targeting in T. brucei (Blundell et al 1996). Recombination during TAR cloning, however, is relatively insensitive to mismatches, as demonstrated by using Alu repeats, which are shorter (∼300 bp) than the target used here (560 bp) and more divergent (85% identity on average) (Larionov et al 1996b;Noskov et al 2003b). The distribution of the non-BES3 clones is similar to that of random telomere targeting in yeast, in which there are imperfect repeats (Louis and Borts 1995).…”

Section: Cloning Bias Of T Brucei Bessmentioning

confidence: 97%

Isolation of the repertoire of VSG expression site containing telomeres of Trypanosoma brucei 427 using transformation-associated recombination in yeast

Becker¹,

Aitcheson²,

Byles³

et al. 2004

Genome Res.

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Trypanosoma brucei switches between variant surface glycoproteins (VSGs) allowing immune escape. The active VSG is in one of many telomeric bloodstream form VSG expression sites (BESs), also containing expression site-associated genes (ESAGs) involved in host adaptation. The role of BES sequence diversity in parasite virulence can best be understood through analysis of the full repertoire of BESs from a given T. brucei strain. However, few BESs have been cloned, as telomeres are highly underrepresented in standard libraries. We devised a strategy for isolating the repertoire of T. brucei 427 BES-containing telomeres in Saccaromyces cerevisiae by using transformation-associated recombination (TAR). We isolated 182 T. brucei 427 BES TAR clones, 167 of which could be subdivided into minimally 17 BES groups. This set gives us the first view of the breadth and diversity of BESs from one T. brucei strain. Most BESs ranged between 40 and 70 kb (average, 57 ± 17 kb) and contained most identified ESAGs. Phylogenetic comparison of the cohort of BES promoter and ESAG6 sequences did not show similar trees, indicating rapid evolution most likely mediated by sequence exchange between BESs. This cloning strategy could be used for any T. brucei strain, facilitating research on the biodiversity of telomeric gene families and host-pathogen interactions.

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“…The highly transformable Saccharomyces cerevisiae strain VL6-48 (MATa, his3-⌬200, trp1-⌬1, ura3-52, lys2, ade2-101, met14) (8), which has HIS3 deleted was used for transformations. Spheroplasts that enable efficient transformation were generated by using a previously described protocol (1).…”

Section: Methodsmentioning

confidence: 99%

“…Spheroplasts that enable efficient transformation were generated by using a previously described protocol (1). Agarose plugs (100 l) containing approximately 5 g of high molecular weight human DNA were prepared from normal human fibroblasts MRC-5 (American Type Culture Collection) and used for yeast transformation (1,8). Linearized TAR cloning vector (1 g) was added to the DNA-containing plugs before treating with agarase and presented to spheroplasts.…”

Section: Methodsmentioning

confidence: 99%

Functional copies of a human gene can be directly isolated by transformation-associated recombination cloning with a small 3′ end target sequence

Kouprina

Annab

Graves

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

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Unique, small sequences (sequence tag sites) have been identified at the 3 ends of most human genes that serve as landmarks in genome mapping. We investigated whether a single copy gene could be isolated directly from total human DNA by transformation-associated recombination (TAR) cloning in yeast using a short, 3 unique target. A TAR cloning vector was constructed that, when linearized, contained a small amount (381 bp) of 3 hypoxanthine phosphoribosyltransferase (HPRT) sequence at one end and an 189-bp Alu repeat at the other end. Transformation with this vector along with human DNA led to selective isolations of the entire HPRT gene as yeast artificial chromosomes (YACs) that extended from the 3 end sequence to various Alu positions as much as 600 kb upstream. These YACs were retrofitted with a Neo R and a bacterial artificial chromosome (BAC) sequence to transfer the YACs to bacteria and subsequently the BACs to mouse cells by using a Neo selection. Most of the HPRT isolates were functional, demonstrating that TAR cloning retains the functional integrity of the isolated material. Thus, this modified version of TAR cloning, which we refer to as radial TAR cloning, can be used to isolate large segments of the human genome accurately and directly with only a small amount of sequence information.

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Highly selective isolation of human DNAs from rodent–human hybrid cells as circular yeast artificial chromosomes by transformation-associated recombination cloning

Cited by 50 publications

References 24 publications

Recovery and potential utility of YACs as circular YACs/BACs

Recovery and potential utility of YACs as circular YACs/BACs

Isolation of the repertoire of VSG expression site containing telomeres of Trypanosoma brucei 427 using transformation-associated recombination in yeast

Functional copies of a human gene can be directly isolated by transformation-associated recombination cloning with a small 3′ end target sequence

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