Cloning of the chick hsp 90 cDNA in expression vector

Abstract

A cDNA clone for the 90kDa heat-shock protein, which we have recently identified as a component of steroid hormone receptors in their heteromeric 8S form, was isolated by direct immunological screening of a chicken smooth muscle cDNA expression library, prepared in the expression plasmids pUC8 and pUC9. Using polyclonal and monoclonal antibodies against the 90kDa protein a colony was identified that reacted with both antibodies. Plasmid 9.11 (p9.11, ˜ 1100 base pair insert) was found to hybrid-select mRNA for the 90kDa heat-shock protein. Northern blot analysis revealed that RNA isolated from various chicken tissues contain a single transcript of ˜ 3 Kb hybridizing to a [³²P]labelled cDNA probe made from p9.11. Heat-shock treatment of chick embryonic fibroblasts resulted in increased steady-state levels of a 3 Kb transcript in both poly A+ and poly A− RNA fractions. Southern blot analysis of chicken genomic DNA indicated that the cDNA hybridizes to a single copy sequence. Sequence data show that the p9.11 cDNA displays a high degree of homology with the 5′ portion of yeast heat shock protein 90 cDNA.