title: A deep coverage Dictyostelium discoideum genomic DNA library replicates stably in Escherichia coli.
abstract: The natural history of the amoeba Dictyostelium discoideum has inspired scientific inquiry for seventy-five years. A genetically tractable haploid eukaryote, D. discoideum appeals as a laboratory model as well. However, certain rote molecular genetic tasks, such as PCR and cloning, are difficult due to the AT-richness and low complexity of its genome. Here we report on the construction of a ~20 fold coverage D. discoideum genomic library in Escherichia coli, cloning 4-10 kilobase partial restriction fragments into a linear vector. End-sequencing indicates that most clones map to the six chromosomes in an unbiased distribution. Over 70% of these clones contain at least one complete open reading frame. We demonstrate that individual clones and library composition are stable over multiple replication cycles. Our library will enable numerous molecular biological applications and the completion of additional species' genome sequences, and suggests a path towards the long-elusive goal of genetic complementation.
authors.affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
authors.forename: Rafael D
authors.initials: RD
authors.lastname: Rosengarten
authors.affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
authors.forename: Pamela R
authors.initials: PR
authors.lastname: Beltran
authors.affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA. Electronic address: gadi@bcm.edu.
authors.forename: Gad
authors.initials: G
authors.lastname: Shaulsky
mesh_terms: D003001:Cloning, Molecular; D004023:Dictyostelium / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D018503:Genome, Protozoan; D015698:Genomic Library; D017422:Sequence Analysis, DNA
keywords: Dictyostelium genomic library; Linear bacteriophage plasmid