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Proc Natl Acad Sci U S A. 1989 Oct;86(20):7966-70.

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

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1
Department of Biology, University of California, San Diego, La Jolla 92093.

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.

PMID:
2813371
PMCID:
PMC298193
[Indexed for MEDLINE]
Free PMC Article
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