Dataset Information

Total synthesis of long DNA sequences: synthesis of a contiguous 32-kb polyketide synthase gene cluster.

ABSTRACT: To exploit the huge potential of whole-genome sequence information, the ability to efficiently synthesize long, accurate DNA sequences is becoming increasingly important. An approach proposed toward this end involves the synthesis of approximately 5-kb segments of DNA, followed by their assembly into longer sequences by conventional cloning methods [Smith, H. O., Hutchinson, C. A., III, Pfannkoch, C. & Venter, J. C. (2003) Proc. Natl. Acad. Sci. USA 100, 15440-15445]. The major current impediment to the success of this tactic is the difficulty of building the approximately 5-kb components accurately, efficiently, and rapidly from short synthetic oligonucleotide building blocks. We have developed and implemented a strategy for the high-throughput synthesis of long, accurate DNA sequences. Unpurified 40-base synthetic oligonucleotides are built into 500- to 800-bp "synthons" with low error frequency by automated PCR-based gene synthesis. By parallel processing, these synthons are efficiently joined into multisynthon approximately 5-kb segments by using only three endonucleases and "ligation by selection." These large segments can be subsequently assembled into very long sequences by conventional cloning. We validated the approach by building a synthetic 31,656-bp polyketide synthase gene cluster whose functionality was demonstrated by its ability to produce the megaenzyme and its polyketide product in Escherichia coli.

SUBMITTER: Kodumal SJ

PROVIDER: S-EPMC524854 | biostudies-literature | 2004 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Total synthesis of long DNA sequences: synthesis of a contiguous 32-kb polyketide synthase gene cluster.

Kodumal Sarah J SJ Patel Kedar G KG Reid Ralph R Menzella Hugo G HG Welch Mark M Santi Daniel V DV

Proceedings of the National Academy of Sciences of the United States of America 20041020 44

To exploit the huge potential of whole-genome sequence information, the ability to efficiently synthesize long, accurate DNA sequences is becoming increasingly important. An approach proposed toward this end involves the synthesis of approximately 5-kb segments of DNA, followed by their assembly into longer sequences by conventional cloning methods [Smith, H. O., Hutchinson, C. A., III, Pfannkoch, C. & Venter, J. C. (2003) Proc. Natl. Acad. Sci. USA 100, 15440-15445]. The major current impedimen ...[more]

PMID: 15496466

Similar Datasets

Project description:Pseudocercospora fijiensis, causal agent of black Sigatoka of banana, produces polyketide synthase (PKS) pathways shown to be important in disease development by related Dothideomycete fungi. Genome analysis of the P. fijiensis PKS8-1 gene identified it as part of a gene cluster including genes encoding two transcription factors, a regulatory protein, a glyoxylase/beta-lactamase-like protein, an MFS transporter, a cytochrome P450, two aldo/keto reductases, a dehydrogenase, and a decarboxylase. Genome analysis of the related pathogens Pseudocercospora musae, Pseudocercospora eumusae, and Pseudocercospora pini-densiflorae, identified orthologous clusters containing a nearly identical combination of genes. Phylogenetic analysis of PKS8-1 identified homology to PKS proteins in the monodictyphenone and cladofulvin pathways in Aspergillus nidulans and Cladosporium fulvum, respectively. Analysis of clustered genes showed that the PKS8-1 cluster shares genes for enzymes involved in the production of the emodin intermediate in the monodictyphenone and cladofulvin pathways, but differs in many genes, suggesting production of a different metabolic product. Time course analysis of gene expression in infected banana showed up-regulation of PKS8-1 and four of eight clustered genes as early as 2 weeks post-inoculation and remaining high through 9 weeks. Overexpression of the pathway through constitutive expression of an aflR-like transcription factor gene in the cluster resulted in increased expression in culture of PKS8-1 as well as the four clustered genes that are up-regulated in infected plants. No differences were seen in timing or severity of disease symptoms with the overexpression strains relative to controls, however gene expression analysis showed no difference in expression in planta by an overexpression strain relative to controls. Thus constitutive expression of the aflR-like gene is not sufficient to upregulate the pathway above normal expression in planta. Pathway expression during all phases of disease development and conservation of the pathway in related Pseudocercospora species support a role for this pathway in disease.

Dataset Information

Total synthesis of long DNA sequences: synthesis of a contiguous 32-kb polyketide synthase gene cluster.

Publications

Total synthesis of long DNA sequences: synthesis of a contiguous 32-kb polyketide synthase gene cluster.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets