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Nucleic Acids Research, 1981, Vol. 9, No. 4 947-963
© 1981

MOLECULAR BIOLOGY

Selection and analysis of cloned developmentally-regulated Dictyostelium discoideum genes by hybridization-competition

Giorgio Mangiarotti1, Stephen Chung, Charles Zuker and Harvey F. Lodish

Department of Biology, Massachusetts Institute of Technology Cambridge, MA 02139, USA

Received October 15, 1980.

We describe a new technique for selection of cloned gene segments which are expressed preferentially at one developmental stage but at a relatively low level. A nitrocellulose filter replica of plaques of {lambda} phage which contain approximately 8 KB inserts of genomic DNA is prepared; it is hybridized with a small amount of [32P] labeled mRNA prepared from one developmental stage, in the presence of a several-hundred fold excess of competitor RNA from a different stage. We show that clones of Dictyostelium nuclear DNA which form hybrids under these conditions indeed encode developmentally regulated mRNAs. Our previous analysis of Dictyostelium discoideum differentiation indicated that transcripts from about 12% of the genome appear in mRNA at one defined stage of differentiation - the formation of cell-cell aggregates. A number of our new clones are novel, in that they encode multiple discrete mRNA species all of which accumulate only at the cell aggregate stages; others encode one or more mRNAs which appear at the tight aggregate stage and also one or more which are present throughout differentiation. These latter clones, in particular, would be difficult to identify using other selection techniques.

1Permanent address: Cattedra di Biologia Generale per Medicina, University of Turin, Italy


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