BIOL2609_L22_2007

BIOL2609_L22_2007 - Molecular Ecology ECOL 2007 Lecture 22:...

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Unformatted text preview: Molecular Ecology ECOL 2007 Lecture 22: Stress responses: Expression of stress protein genes The heat shock response • The heat shock response is a ubiquitous, protective (and homeostatic) cellular response to cope with heat-induced damage to proteins • Many ‘heat shock proteins’ (HSP’s) are produced as a response – These can be molecular chaperones, or ATP-dependant proteases • They function in protein folding, repair and degradation (under both normal and stress conditions) Heat shock Co-factors activated e.g. stabilization of usually unstable molecules, melting of mRNA structures Feedback control Translation of heat shock proteins which act to protect cellular proteins from heat damage Binding to promoter sequences of sigma factor (prokaryotes) or heat shock response elements (eukaryotes) Heat shock protein gene transcription Heat-shock response in the antarctic ciliate Euplotes • Eukaryotic microbial diversity is high in Antarctic waters • Protozoan ciliates, particularly of the genus Euplotes, are very common • There are 3 species endemic to the Antarctic: – E.euryhalinus – E.focardii – E.nobilii • All are found in freezing (-1.8C) benthic interstitial water In lab culture their response to warming (heat shock) varies: • E.euryhalinus & E.nobilii display psychrotrophic behaviour – They multiply faster at 12-18C rather than 4C • Conversely, E.focardii displays distinct psychrophilic behaviour – Sharp decline in survival and multiplication at 8-10C – Irreversible cell damage above 20C • Molecular techniques were used to assess the molecular basis for this difference in response to thermal stress • In this study: – HSP genes in nuclear DNA identified by southern blot – HSP mRNA transcription measured by northern blot • A universally conserved heat shock protein, HSP 70, was studied in E.nobilii (En) (psychrotroph) and E.focardii (Ef) (psychrophile) HSP 70 gene identification • Nuclear DNA extracted using standard methods • 1.4kb fragment of HSP 70 gene radio-labelled and used as an oligonucleotide probe in southern blot analysis – – – – Fractionation by electrophoresis Transfer to membrane Wash membrane with radiolabelled probe X-ray film visualization Southern blot identification of HSP 70 genes in Euplotes species Each lane loaded with identical amounts of DNA (8ug) (E.raikovi is a nonantarctic species from which the oligo probe was obtained) • Southern blot reveals: – Ef band at 2.5kb – En band at 2.4kb • Higher band intensity in Ef implies – Greater sequence homology – Massive copy number (Er known to have several thousand in genome) HSP 70 gene transcription Heat shock exposure: • Cells grown at ‘standard ‘ temperature (4C), followed by • ‘Abrupt’ increases (8, 12, 15, 18C) to 20 or 25C • ‘Gradual’ increases to 20 or 25C • Total mRNA was extracted from cell lysates – – – – Fractionated by electrophoresis Transferrred to membrane Er HSP 70 probe used in northern blot X-ray film visualization Northern blot analysis of total RNA from En and Ef using Er oligo- probe A = HSP 70 probe B = 17S rRNA gene control probe Large gel = abrupt heat shock Small gel = gradual heat shock Arrows = hybridization bands • Regardless of heat shock protocol, En produced a 2.1kb band – This is within the molecular size range expected for HSP 70 • No hybridization signal from Ef could be found, even after 2-3 hours at 20 or 25C • Hybridization signal consistency with the 17S rRNA probe suggests the differences observed for HSP 70 hybridizations reflect transcription and not RNA or reaction quality/quantity • Next, it was assessed whether the observed genetic unresponsiveness of Ef to heat shock was associated with a constitutive inability to express its hsp70 genes • PolyA+ RNA extracted from total RNA of unshocked/shocked cells (I.e. processed gene products, without other RNA’s) to yield RNA 25x more concentrated than in the previous northern blot experiment • Very small amount of HSP 70 transcript were present in non-heat-shocked cells of both Ef and En • Analysis of band intensity showed 70x increase in transcription by En in response to heat shockEf transcription increased only 2x, probably due to increased enzyme efficiency rather than increased transcription • Further support for these results was obtained by analysing protein products in the cells – – – – Protein electrophoresed (40ug/lane) and blotted HSP 70 monoclonal antibodies B-tubulin antibodies (control) Chemiluminescence detection HSP 70 B-tubulin • 70kD protein product consistent with HSP expected size • Band in En 32x more intense than others – control data for B-tubulin support that this is due to greater translation product number in cells Ecological relevance • Why has Ef dramatically reduced or suppressed the response of its HSP 70 genes to heat shock? – Adaptation to highly defined themostable (variation <0.2C) niche ...
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This note was uploaded on 03/19/2010 for the course DEB BIOL2609 taught by Professor Drstephenb.pointing during the Spring '10 term at HKU.

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BIOL2609_L22_2007 - Molecular Ecology ECOL 2007 Lecture 22:...

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