Study of the expression of heat shock genes hsp90, hsp60 and hsp10 of the aquatic fungus Blastocladiella emersonii

Author(s)

Luciana Pugliese da Silva

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ)
• Defense date: 2003-01-08

Examining board members

Suely Lopes Gomes; Luis Eduardo Soares Netto; Carlos Henrique Inacio Ramos; Roberto Vicente Santelli; Aline Maria da Silva

• Advisor: Suely Lopes Gomes

Abstract

The heat shock protein 90 (Hsp90) is a cytosolic molecular chaperone. The incomplete cDNA of this protein was isolated by immunoblot screening of a heat shock cDNA expression library. The complete genomic clone was also isolated and completely sequenced and characterized. The coding sequence is interrupted by a single intron with 184 nucleotides. The deduced amino acid sequence corresponds to a 710-residue polypeptide with a calculated molecular mass of 80,792 Da and an average pl of 4.85. Primer extension and RACE-PCR experiments demonstrated a single transcription start site localized -65 and -70 nucleotides from de ATG of the initiator methionine, respectively. Sequence motifs resembling the standard eukaryotic heat shock element (HSE) and the stress responsive element (STRE) were evident in the regulatory region -395 and -98 nucleotides from de ATG, respectively. Northern blot analysis revealed that the Hsp90 mRNA presents maximum levels by 90 minutes of the sporulation stage. Immunoblot analysis indicated that the Hsp90 is present during the entire life cycle of the fungus and maximum levels were observed 90 minutes after the induction of sporulation, indicating a transcriptional control. During heat shock both the mRNA and the Hsp90 protein are highly induced. Proteins Hsp60 and Hsp10, are mitochondrial molecular chaperones (chaperonines). The complete cDNAs encoding these proteins were and completely sequenced. The deduced amino acid sequence for Hsp60 corresponds to a 559-residue polypeptide with a calculated molecular mass of 58,741 Da and an average pl of 8.7. Immunoblot analysis showed that Hsp60 is present during the entire life cycle of the fungus and presents maximum levels by 90 minutes of the sporulation. Northern blot analysis indicated maximum levels of the Hsp60 mRNA by 90 minutes of sporulation too. Both mRNA and the protein are highly induced during heat shock. The deduced amino acid sequence for Hsp10 corresponds to a 101-residue polypeptide with a calculated molecular mass of 10,688 Da and an average pl of 6.25. Northern blot analysis indicated maximum mRNA levels by 120 minutes of germination and high levels of expression when the cells are exposed to heat shock. (AU)