Abstract
Extracellular cAMP serves as a chemoattractant as well as a signal which regulates gene expression during development of Dictyostelium discoideum. The cell adhesion molecule gp80 is expressed at the aggregation stage, between 6 and 10 h of development, and is known to be under cAMP regulation. Transcription of the gp80 gene is first turned on at a low, basal level at the preaggregation stage and is then greatly augmented by pulses of low levels of cAMP at the aggregation stage. Using cloned cDNA sequences, we have isolated genomic DNA fragments encompassing the gp80 gene. The gp80 gene has a single open reading frame, with multiple transcription start sites located downstream from a putative TATA box. Several short, repeated sequences in the upstream sequence have also been identified. The cloned 1.3-kilobase upstream DNA was sufficient to confer proper temporal and cAMP regulation on a gp80 minigene reporter in Dictyostelium cells. Deletional analysis of this 5'-flanking DNA led to the mapping of a cAMP-response element (CRE) in the gp80 gene to sequences between -306 and -289 base pairs upstream of the translational start site. Present within this region is a sequence we refer to as box 1 (TGGTGTG). The gp80 box 1-CRE binds specifically to a protein present in nuclear extracts, but binding is abolished when mutations are introduced in the box 1 sequence. The gp80 box 1-CRE shows little sequence homology to CREs of late developmental genes and the expression of gp80 may involve a distinct signal transduction pathway.
Highlights
The preaggregationstageandisthengreatlyaugmented by pulses of low levels of cAMP at the aggregation stage
DNA led to the mapping of a CAMP-response element cumulate rapidly on the cell surface between 6 and 10 h of (CRE) in the gpS0 gene to sequences between -306 development, corresponding tothe aggregation stage [16]
CDNA clones for gp80 have been isolated and used in the study of the expression of gp80(18-21).gp80 is similar to several cAMP pulse-induced genes in that itappears to have a two-phase regulatory pathway [10,22]
Summary
Cell Strains and Culture Conditions-The axenic strains AX2 and tor-For the wild-type box 1 oligonucleotides (bl), two synthetic. The oligonucleotides containing two Purification of DNA from D. discoideum-AX2 cells were lysed in copies of box 4 (b4-2) are similar except the sequence from -662 to. By lysing the cells with 10% sodium dodecyl sulfate,10%diethyl and the inserwtsere subcloned into pEMBL18for restriction mapping pyrocarbonate The Either the 24-bp box 1-oligonucleotide ( b l ) described above, or a ends were treated with the Klenowenzyme, HindIII linkers were 40-bp box 1-containing fragment (corresponding to gp sequences added, and HindIII-BamHI fragments were isolated and subcloned from -325 to -289 bp), was labeled with[w3*P]dATP using the back into the pBl0TPl vector.
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