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PMID: 2813371 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

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

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 86 ·No. 20 ·1989-10-00 ·Pages 7966-70

Dynes JL, Firtel RA

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.

MeSH Terms
Amino Acid Sequence Base Sequence DNA, Fungal/genetics Dictyostelium/genetics,physiology Electric Stimulation/methods Genetic Complementation Test Genetic Markers/analysis Genetic Vectors Genomic Library Information Systems Molecular Sequence Data Mutation Plasmids RNA, Fungal/genetics Restriction Mapping Thymidine/metabolism
Chemicals
DNA, Fungal Genetic Markers RNA, Fungal Thymidine
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Dynes J L
Department of Biology, University of California, San Diego, La Jolla 92093.
Firtel R A
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Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.86.20.7966
Published
1989-10-00
Pages
7966-70
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC298193
Subset
IM
Databases
GENBANK
M27713
External Links
PubMed source DOI: 10.1073/pnas.86.20.7966 PMC Full Text View journal details
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