Abstract
The genomic structure of the alpha-actinin gene from Dictyostelium discoideum has been determined. The coding region consists of three exons separated by two short introns located at either end of the gene. In an alpha-actinin mutant, HG1130, about 1% of alpha-actinin protein products are found compared to the parent strain AX2. The size of the mutant proteins are 88 and 95 kDa. In HG1130, the second intron is improperly spliced from the primary alpha-actinin transcript resulting from a mutation at the 5' splice site from a GT to an AT. The premature RNA is less efficiently cleaved at the mutated 5' splice site and the following step of the splicing reaction, ligation of exons 2 and 3, is prevented. Thus, two RNA species are generated, one in which intron 2 is not removed and one in which exon 2 is not ligated to exon 3. The two RNA species are stable, transported to the cytoplasm, bound to polysomes and translated. In vitro and in vivo, the partially spliced RNAs are translated into proteins of 88 and 95 kDa which can be immunoprecipitated with an alpha-actinin-specific monoclonal antibody. The in vivo stability of the mutant proteins is comparable to the wild type alpha-actinin.
Highlights
A Single Base Exchange in an Intron of the Dictyostelium discoideum cu-Actinin Gene Inhibits Correct Splicing of the RNA but Allows Transport to the Cytoplasm and Translation*
Genomic Organization of the D. discoideum a-Actinin Gene-In D. discoideum cy-actinin is encoded by a single gene (Witke et al, 1986). cr-Actinin is an F-actin cross-linking protein present in most muscle and non-muscle cells
Dimerization, and Ca2+ regulation are explained in a model derived from the cDNA sequence of D. discoideum a-actinin (Noegel et al, 1987)
Summary
After 5 min of heat denaturation the samples were incubated at room temperature for 30 min to anneal the primer to the RNA template. SDS, 2 mM EDTA, 120 mM sodium phosphate, pH 6.8. SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; bp, base pair(s); PCR, polymerase chain reaction; PIPES, 1,4-piperazinediethanesulfonic acid. Fragment of the a-actinin gene has been published previously (Witke et al, 1986). C-terminal part of the HG1130 a-actinin gene, spanning a 1122-bp region from the EcoRI to the PuuII site was amplified through the polymerase chain reaction with the heat-stable. To facilitate cloning of the amplified DNA, an EcoRI site in oligonucleotide
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