Segments missing from the draft human genome sequence can be isolated by transformation‐associated recombination cloning in yeast

Kouprina, Natalay; Leem, Sun‐Hee; Solomon, Greg; Ly, Albert; Koriabine, Maxim; Otstot, John; Pak, Eugene; Dutra, Amalia; Zhao, Shaying; Barrett, J. Carl; Larionov, Vladimir

doi:10.1038/sj.embor.embor766

Cited by 37 publications

(36 citation statements)

References 22 publications

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“…Thus, we turned to S. cerevisiae as a cloning host. Yeast will support at least 2 Mb of DNA in a linear centromeric yeast artificial chromosome (YAC) (15) and has been used to clone sequences that are unstable in E. coli (16).…”

Section: Figmentioning

confidence: 99%

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Complete Chemical Synthesis, Assembly, and Cloning of a Mycoplasma genitalium Genome

Gibson

Benders

Andrews-Pfannkoch

et al. 2008

Science

1,110

835

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We have synthesized a 582,970-base pair Mycoplasma genitalium genome. This synthetic genome, named M. genitalium JCVI-1.0, contains all the genes of wild-type M. genitalium G37 except MG408, which was disrupted by an antibiotic marker to block pathogenicity and to allow for selection. To identify the genome as synthetic, we inserted "watermarks" at intergenic sites known to tolerate transposon insertions. Overlapping "cassettes" of 5 to 7 kilobases (kb), assembled from chemically synthesized oligonucleotides, were joined by in vitro recombination to produce intermediate assemblies of approximately 24 kb, 72 kb ("1/8 genome"), and 144 kb ("1/4 genome"), which were all cloned as bacterial artificial chromosomes in Escherichia coli. Most of these intermediate clones were sequenced, and clones of all four 1/4 genomes with the correct sequence were identified. The complete synthetic genome was assembled by transformation-associated recombination cloning in the yeast Saccharomyces cerevisiae, then isolated and sequenced. A clone with the correct sequence was identified. The methods described here will be generally useful for constructing large DNA molecules from chemically synthesized pieces and also from combinations of natural and synthetic DNA segments.

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Section: Figmentioning

confidence: 99%

“…Some evidence for asymmetric explosions was obtained from the polarization detected in several SNe II (12) and SNe Ib/c (13) and in the broad-lined SN Ic 2002ap (14)(15)(16). However, because there are still few such detections, no systematic study exists.…”

mentioning

confidence: 92%

Complete Chemical Synthesis, Assembly, and Cloning of a Mycoplasma genitalium Genome

Gibson

Benders

Andrews-Pfannkoch

et al. 2008

Science

1,110

835

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“…Therefore, we cloned and used a Ն54-kb genomic region containing all exons and introns and at least 11-kb upstream and 1.2-kb downstream sequences of the hTERT gene (17). Whereas the absence or instability of some human DNA sequences in conventional genomic libraries has hampered studies on functions and regulations of an entire gene locus and has left a number of gaps in the human genome sequence database, transformation-associated recombination (TAR) cloning enables the efficient, reproducible cloning and stable maintenance of large genomic regions of interest and, thus, is a powerful approach to solve these problems in genome research (31,32). This study presents a successful application of an entire human gene isolated by TAR cloning in in vivo analysis of gene regulation.…”

Section: Discussionmentioning

confidence: 99%

Differential cis-regulation of human versus mouse TERT gene expression in vivo : Identification of a human-specific repressive element

Horikawa

Chiang

Patterson

et al. 2005

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

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In vivo expression of human telomerase is significantly different from that of mouse telomerase. To assess the basis for this difference, a bacterial artificial chromosome clone containing the entire hTERT (human telomerase reverse transcriptase) gene was introduced in mice. In these transgenic mice, expression of the hTERT transgene was similar to that of endogenous hTERT in humans, rather than endogenous mTERT (mouse telomerase reverse transcriptase). In tissues and cells showing a striking difference in expression levels between hTERT in humans and mTERT in mice (i.e., liver, kidney, lung, uterus, and fibroblasts), expression of the hTERT transgene in transgenic mice was repressed, mimicking hTERT in humans. The transcriptional activity of the hTERT promoter was much lower than that of the mTERT promoter in mouse embryonic fibroblasts or human fibroblasts. Mutational analysis of the hTERT and mTERT promoters revealed that a nonconserved GC-box within the hTERT promoter was responsible for the humanspecific repression. These results reveal that a difference in cisregulation of transcription, rather than transacting transcription factors, is critical to species differences in tissue-specific TERT expression. Our data also suggest that the GC-box-mediated, human-specific mechanism for TERT repression is impaired in human cancers. This study represents a detailed characterization of the functional difference in a gene promoter of mice versus humans and provides not only important insight into speciesspecific regulation of telomerase and telomeres but also an experimental basis for generating mice humanized for telomerase enzyme and its pattern of expression.

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“…The "finished" human sequence still contains a number of gaps that can be distinguished in "sequence gaps" when spanned by one or more sequencing clones, and "clone gaps" if they separate neighboring contigs (Eichler et al 2004;Kouprina et al 2003;Leem et al 2004).…”

Section: Introductionmentioning

confidence: 99%

A satellite-like sequence, representing a “clone gap” in the human genome, was likely involved in the seeding of a novel centromere in macaque

Carbone¹,

D’Addabbo

Cardone

et al. 2008

Chromosoma

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Although the human genome sequence is generally considered "finished", the latest assembly (NCBI Build 36.1) still presents a number of gaps. Some of them are defined as "clone gaps" because they separate neighboring contigs. Evolutionary new centromeres are centromeres that repositioned along the chromosome, without marker order variation, during evolution. We have found that one human "clone gap" at 18q21.2 corresponds to an evolutionary new centromere in Old World Monkeys (OWM). The partially sequenced gap revealed a satellite-like structure. DNA stretches of the same satellite were found in the macaque (flanking the chromosome 18 centromere) and in the marmoset (New World Monkey), which was used as an outgroup. These findings strongly suggested that the repeat was present at the time of novel centromere seeding in OWM ancestor. We have provided, therefore, the first instance of a specific sequence hypothesized to have played a role in triggering the emergence of an evolutionary new centromere.

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Segments missing from the draft human genome sequence can be isolated by transformation‐associated recombination cloning in yeast

Cited by 37 publications

References 22 publications

Complete Chemical Synthesis, Assembly, and Cloning of a Mycoplasma genitalium Genome

Complete Chemical Synthesis, Assembly, and Cloning of a Mycoplasma genitalium Genome

Differential cis-regulation of human versus mouse TERT gene expression in vivo : Identification of a human-specific repressive element

A satellite-like sequence, representing a “clone gap” in the human genome, was likely involved in the seeding of a novel centromere in macaque

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