Cloning of the chick hsp 90 cDNA in expression vector

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• The 90kDa heat-shock protein (HSP90) is recognized as a component of steroid hormone receptors in their 8S heteromeric form.
• Understanding HSP90's molecular characteristics is vital for comprehending steroid hormone receptor function.

Purpose of the Study:

• To isolate and characterize a cDNA clone encoding the chicken 90kDa heat-shock protein.
• To investigate the expression and genomic organization of the HSP90 gene in chickens.

Main Methods:

• Direct immunological screening of a chicken smooth muscle cDNA expression library using polyclonal and monoclonal antibodies against HSP90.
• Hybrid selection of mRNA and Northern blot analysis to confirm the identity and transcript size of the HSP90 cDNA.
• Southern blot analysis to determine the gene copy number and sequence homology analysis with yeast HSP90 cDNA.

Main Results:

• A cDNA clone (p9.11) encoding the chicken 90kDa heat-shock protein was successfully isolated.
• Northern blot analysis confirmed a single 3 Kb transcript for HSP90 in various chicken tissues, with increased levels upon heat shock.
• Southern blot analysis indicated that the HSP90 cDNA represents a single-copy sequence in the chicken genome.
• Sequence data revealed significant homology between the chicken HSP90 cDNA and the 5' portion of yeast HSP90 cDNA.

Conclusions:

• The isolated cDNA clone represents the chicken 90kDa heat-shock protein, a key component of steroid hormone receptors.
• The expression of HSP90 mRNA is regulated by heat shock, suggesting a conserved stress response mechanism.
• High homology with yeast HSP90 suggests conserved structural and functional domains across species.