A Model System to Assess the Integrity of Mammalian YACs during Transformation and Propagation in Yeast

411

318

We previously reported assembly and cloning of the synthetic Mycoplasma genitalium JCVI-1.0 genome in the yeast Saccharomyces cerevisiae by recombination of six overlapping DNA fragments to produce a 592-kb circle. Here we extend this approach by demonstrating assembly of the synthetic genome from 25 overlapping fragments in a single step. The use of yeast recombination greatly simplifies the assembly of large DNA molecules from both synthetic and natural fragments.in vivo DNA assembly ͉ genome synthesis ͉ combinatorial assembly ͉ yeast transformation ͉ Mycoplasma genitalium ͉ synthetic biology Y east has long been considered a genetically tractable organism because of its ability to take up and recombine DNA fragments. More than 30 years ago, Hinnen et al. (1) reported the restoration of leucine biosynthesis in Saccharomyces cerevisiae by transformation of a leu2 -strain to LEU2ϩ using a method involving spheroplasts, CaCl 2 , and PEG. Soon after, Orr-Weaver et al. (2) reported mechanistic studies demonstrating that DNA molecules taken up during yeast transformation can integrate into yeast chromosomes through homologous recombination, and that the ends of the linear-transforming DNA are highly recombinogenic and react directly with homologous chromosomal sequences, whereas circular plasmids carrying yeast sequences integrate by a single crossover and only at low frequency. Subsequently, yeast transformation has become an indispensable tool in yeast genetics.Yeast recombination has since been applied to the construction of plasmids and yeast artificial chromosomes (YACs). In 1987, Ma et al. (3) constructed plasmids from two cotransformed DNA fragments containing homologous regions. In another process, called linker-mediated assembly, any DNA sequence can be joined to a vector DNA using short synthetic linkers that bridge the ends (4, 5). Similarly, four or five overlapping DNA pieces can be assembled and joined to vector DNA (4, 6, 7). This work demonstrated that yeast cells can take up multiple pieces of DNA, and that homologous yeast recombination is sufficiently efficient to correctly assemble the pieces into a single recombinant molecule.The limitations of assembly methods in yeast remain unknown. We recently assembled an entire synthetic M. genitalium genome using a combination of in vitro enzymatic recombination in early stages and in vivo yeast recombination in the final stage to produce the complete genome (8). In the first stage, overlapping Ϸ6-kb DNA cassettes were joined four at a time to form 25 Ϸ24-kb A-series assemblies. Three A-series assemblies were then joined to make 1/8 genome Ϸ72-kb B-series assemblies, and then two Bseries assemblies were assembled to make each of the Ϸ145-kb quarter-genome C-series assemblies. We accomplished the final assembly in yeast using three quarter-genome fragments and a fourth quarter fragment that had been cleaved by a restriction enzyme to provide a site for insertion of the vector DNA. Thus, some individual yeast cells have the capacity to simultaneously tak...

Section: Discussionmentioning

confidence: 99%

One-step assembly in yeast of 25 overlapping DNA fragments to form a complete synthetic Mycoplasma genitalium genome

Gibson

Benders

Axelrod

et al. 2008

411

318

“…Radiation hybrid hamsterhuman cell line D2-X-38 that contained an approximately 5-Mb fragment from chromosome 2 with the XRCC5 (Ku80) DNA repair gene (11) was obtained from P. Jeggo (University of Sussex, Sussex, U.K.). Yeast cells were grown on complete medium (YEPD) or synthetic standard selective media (14).…”

Section: Tar Cloning Vectors Centromeric and Acentric Vectors Pvc1mentioning

confidence: 99%

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

Larionov

Kouprina

Graves

et al. 1996

Transformation-associated recombination (TAR) can be exploited in yeast to clone human DNAs. TAR cloning was previously accomplished using one or two telomere-containing vectors with a common human repeat(s) that could recombine with human DNA during transformation to generate yeast artificial chromosomes (YACs). On basis of the proposal that broken DNA ends are more recombinogenic than internal sequences, we have investigated if

“…However, artifacts such as chimeras and deletions can limit their use (5)(6)(7)(8). These artifacts may result from in vitro DNA manipulation resulting in the DNA becoming broken or nicked.…”

mentioning

confidence: 99%

“…The vector pVL27 was constructed by ligating the 2.2-kb Pvu I fragment from pBP109 with a 2.5-kb Pvu I fragment from pRS306 (16 16 was provided by L. Deaven, Los Alamos National Laboratory. Yeast cells were grown on complete medium (YPD) or synthetic standard selective media (5).…”

mentioning

confidence: 99%

Specific cloning of human DNA as yeast artificial chromosomes by transformation-associated recombination.

Larionov

Kouprina

Graves

et al. 1996

146

132

DNA molecules undergoing transformation into yeast are highly recombinogenic, even when diverged. We reasoned that transformation-associated recombination (TAR) could be employed to clone large DNAs containing repeat sequences, thereby eliminating the need for in vitro enzymatic reactions such as restriction and ligation and reducing the amount of DNA handling. Gently isolated human DNA was transformed directly into yeast spheroplasts along with two genetically marked (Ml and M2) linearized vectors that contained a human Alu sequence at one end and a telomere sequence at the other end (Alu-CEN-MI-TEL and Alu-M2-TEL). Nearly all the Mi-selected transformants had yeast artificial chromosomes (YACs) containing human DNA inserts that varied in size from 70 kb to >600 kb. Approximately half of these had also acquired the unselected M2 marker. The mitotic segregational stability of YACs generated from one (Ml) or two (Ml and M2) vector(s) was comparable, suggesting de novo generation of telomeric ends. Since no YACs were isolated when rodent DNAs or a vector lacking an Alu sequence was used, the YACs were most likely the consequence of TAR between the repeat elements on the vector(s) and the human DNA.