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Molecular complementation of a genetic marker in Dictyostelium using a genomic DNA library.

ABSTRACT: We constructed a partial Sau3A Dictyostelium genomic DNA library in a shuttle vector that replicates extrachromosomally in Dictyostelium cells. This library was used to complement Dictyostelium strain HPS400, which lacks thymidylate synthase activity and requires exogenous thymidine for growth. We have used a modified high-frequency transformation protocol that allows the introduction of the library into a sufficient number of Dictyostelium cells to select complementing plasmids. Using this approach, we have isolated a gene (Thy1) that complements the thymidine growth requirement of HPS400. The gene encodes a 1.2-kilobase RNA and the derived amino acid sequence shows no homology to thymidylate synthase, a protein highly conserved throughout evolution, or any other protein sequence in the data base examined. Thy1 provides an important selectable marker for transforming Dictyostelium cells. In addition, this work suggests that it will be possible to isolate genes that are essential for developmental processes in Dictyostelium by complementation.

SUBMITTER: Dynes JL

PROVIDER: S-EPMC298193 | biostudies-other | 1989 Oct

REPOSITORIES: biostudies-other

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Molecular complementation of a genetic marker in Dictyostelium using a genomic DNA library.

Dynes J L JL Firtel R A RA

Proceedings of the National Academy of Sciences of the United States of America 19891001 20

We constructed a partial Sau3A Dictyostelium genomic DNA library in a shuttle vector that replicates extrachromosomally in Dictyostelium cells. This library was used to complement Dictyostelium strain HPS400, which lacks thymidylate synthase activity and requires exogenous thymidine for growth. We have used a modified high-frequency transformation protocol that allows the introduction of the library into a sufficient number of Dictyostelium cells to select complementing plasmids. Using this appr ...[more]

PMID: 2813371

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Project description:Background:Several high-throughput molecular genetic analyses rely on high-quality genomic DNA. Copurification of other molecules can negatively impact the functionality of plant DNA preparations employed in these procedures. Isolating DNA from agronomically important crops, such as sugarcane, rice, citrus, potato and tomato is a challenge due to the presence of high fiber, polysaccharides, or secondary metabolites. We present a simplified, rapid and reproducible SDS-based method that provides high-quality and -quantity of DNA from small amounts of leaf tissue, as required by the emerging biotechnology and molecular genetic applications. Results:We developed the TENS-CO method as a simplified SDS-based isolation procedure with sequential steps of purification to remove polysaccharides and polyphenols using 2-mercaptoethanol and potassium acetate, chloroform partitioning, and sodium acetate/ethanol precipitation to yield high-quantity and -quality DNA consistently from small amounts of tissue (0.15 g) for different plant species. The method is simplified and rapid in terms of requiring minimal manipulation, smaller extraction volume, reduced homogenization time (20 s) and DNA precipitation (one precipitation for 1 h). The method has been demonstrated to accelerate screening of large amounts of plant tissues from species that are rich in polysaccharides and secondary metabolites for Southern blot analysis of reporter gene overexpressing lines, pathogen detection by quantitative PCR, and genotyping of disease-resistant plants using marker-assisted selection. Conclusion:To facilitate molecular genetic studies in major agronomical crops, we have developed the TENS-CO method as a simple, rapid, reproducible and scalable protocol enabling efficient and robust isolation of high-quality and -quantity DNA from small amounts of tissue from sugarcane, rice, citrus, potato, and tomato, thereby reducing significantly the time and resources used for DNA isolation.

Dataset Information

Molecular complementation of a genetic marker in Dictyostelium using a genomic DNA library.

Publications

Molecular complementation of a genetic marker in Dictyostelium using a genomic DNA library.

Similar Datasets

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