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A comprehensive genome-wide map of autonomously replicating sequences in a naive genome.

ABSTRACT: Eukaryotic chromosomes initiate DNA synthesis from multiple replication origins. The machinery that initiates DNA synthesis is highly conserved, but the sites where the replication initiation proteins bind have diverged significantly. Functional comparative genomics is an obvious approach to study the evolution of replication origins. However, to date, the Saccharomyces cerevisiae replication origin map is the only genome map available. Using an iterative approach that combines computational prediction and functional validation, we have generated a high-resolution genome-wide map of DNA replication origins in Kluyveromyces lactis. Unlike other yeasts or metazoans, K. lactis autonomously replicating sequences (KlARSs) contain a 50 bp consensus motif suggestive of a dimeric structure. This motif is necessary and largely sufficient for initiation and was used to dependably identify 145 of the up to 156 non-repetitive intergenic ARSs projected for the K. lactis genome. Though similar in genome sizes, K. lactis has half as many ARSs as its distant relative S. cerevisiae. Comparative genomic analysis shows that ARSs in K. lactis and S. cerevisiae preferentially localize to non-syntenic intergenic regions, linking ARSs with loci of accelerated evolutionary change.

SUBMITTER: Liachko I

PROVIDER: S-EPMC2869322 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

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A comprehensive genome-wide map of autonomously replicating sequences in a naive genome.

Liachko Ivan I Bhaskar Anand A Lee Chanmi C Chung Shau Chee Claire SC Tye Bik-Kwoon BK Keich Uri U

PLoS genetics 20100513 5

Eukaryotic chromosomes initiate DNA synthesis from multiple replication origins. The machinery that initiates DNA synthesis is highly conserved, but the sites where the replication initiation proteins bind have diverged significantly. Functional comparative genomics is an obvious approach to study the evolution of replication origins. However, to date, the Saccharomyces cerevisiae replication origin map is the only genome map available. Using an iterative approach that combines computational pre ...[more]

PMID: 20485513

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Project description:The methylotrophic yeast Pichia pastoris is widely used to produce recombinant proteins, taking advantage of this species' high-density cell growth and strong ability to secrete proteins. Circular plasmids containing the P. pastoris-specific autonomously replicating sequence (PARS1) permit transformation of P. pastoris with higher efficiency than obtained following chromosomal integration by linearized DNA. Unfortunately, however, existing autonomously replicating plasmids are known to be inherently unstable. In this study, we used transcriptome sequencing (RNA-seq) data and genome sequence information to independently identify, on each of the four chromosomes, centromeric DNA sequences consisting of long inverted repeat sequences. By examining the chromosome 2 centromeric DNA sequence (Cen2) in detail, we demonstrate that an ∼111-bp region located at one end of the putative centromeric sequence had autonomous replication activity. In addition, the full-length Cen2 sequence, which contains two long inverted repeat sequences and a nonrepetitive central core region, is needed for the accurate replication and distribution of plasmids in P. pastoris Thus, we constructed a new, stable, autonomously replicating plasmid vector that harbors the entire Cen2 sequence; this episome facilitates genetic manipulation in P. pastoris, providing high transformation efficiency and plasmid stability.IMPORTANCE Secretory production of recombinant proteins is the most important application of the methylotrophic yeast Pichia pastoris, a species that permits mass production of heterologous proteins. To date, the genetic engineering of P. pastoris has relied largely on integrative vectors due to the lack of user-friendly tools. Autonomously replicating Pichia plasmids are expected to facilitate genetic manipulation; however, the existing systems, which use autonomously replicating sequences (ARSs) such as the P. pastoris-specific ARS (PARS1), are known to be inherently unstable for plasmid replication and distribution. Recently, the centromeric DNA sequences of P. pastoris were identified in back-to-back studies published by several groups; therefore, a new episomal plasmid vector with centromere DNA as a tool for genetic manipulation of P. pastoris is ready to be developed.

Dataset Information

A comprehensive genome-wide map of autonomously replicating sequences in a naive genome.

Publications

A comprehensive genome-wide map of autonomously replicating sequences in a naive genome.

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