Molecular Bio Wk 3

What are gene promoters?

-core, distal and proximal promoter regions -can extend 1000s of bp upstream of transcription start site (TSS) -transcription factors binds to promoter elements -activators or repressors bind to regulatory sequences in promoter (enhancer or silencer sequences)

What are methods of isolating RNA?

-solution based -column based

What is solution based methods?

salt and organic solvents used to separate RNA from DNA and protein

What is column based solution?

-RNA like DNA will bind to silica columns under certain conditions -contaminants washed away -RNA eluted from column

What do we use to measure RNA?

-RNA concentration -spectrophotometry (A260/A280) -fluorometry -electrophoresis

Why do we measure RNA?

-used to measure gene expression -aberrant gene expression associated with disease (e.g. cancer) -detect presence of pathogens

How do we measure RNA?

-PCR will only amplify DNA (CANNOT be used for RNA) -reverse transcription (usually followed by PCR (RT-PCR)

What are methods for measuring RNA?

-northern blotting -gel electrophoresis followed by: blotting onto membrane, hybridisation to labelled probe

What is reverse transcription?

-converts RNA to DNA -requires primer or series of small primers -reverse transcription synthesises DNA from template single-stranded RNA molecule -reverse transcription usually following by PCR (RT-PCR)

What is an absolute quantitation example?

-standard curve of known samples -plot graph of (# target DNA molecules(x-axis), log scale) -read off test sample Ct values to determine # of target DNA molecules in sample

What is a relative quantitation example?

-look at the difference in Ct values between test gene and ref gene -look at difference in Ct between sample A and sample B (A = control, B = treatment)

What are Metallothioneins?

-cysteine rich, low molecular weight proteins -bind divalent heavy metal ions -copper, zinc, cadmium, mercury -protect cells from oxidase stress and metal toxicity -found in animals, plants, fungi, bacteria

What happens in Metallothionein gene regulation?

-gene regulation -MTF-1 metal-responsive transcription factor 2 - activator -binds to metal response elements (MREs) in promoter of MT genes - enhancer