WorksheetsAnalysis of Gene Expression
Total questions: 11
Worksheet time: 14mins
Two points concerning the measurements of mRNA levels:
1. the amount of a specific mRNA in a cell at a given point in time is influenced by:
• The level of transcriptional activity
• The stability of that mRNA
2. the amount of mRNA present does not necessarily correlate with the amount of protein made.
1. the amount of a specific mRNA in a cell at a given point in time is influenced by:
• The level of translational activity
• The stability of that mRNA
2. the amount of mRNA present is necessarily correlate with the amount of protein made.
Northern blots is
a technique used to detect specific DNA assuming that the probe we use is sufficiently specific, and to estimate its size.
a technique used to detect specific RNA assuming that the probe we use is sufficiently specific, and to estimate its size.
Limitations of this Northern blots technique:
- not very sensitive, and requires small amounts of RNA (normally at least a microgram of poly(A)-enriched mRNA or 10 μg of total RNA).
- generally not possible to obtain an absolute measurement of the number of molecules of a specific mRNA
- not very sensitive, and requires fairly large amounts of RNA (normally at least a microgram of poly(A)-enriched mRNA or 10 μg of total RNA).
- generally possible to obtain an absolute measurement of the number of molecules of a specific mRNA
In situ hybridization
Visualize the location of specific nucleic acid sequences (gene) within cells or tissues
As an alternative to radioactive labelling, the probe in In situ hybridization can be labelled by:
attaching a fluorescent dye
Methods for studying the promoter
need to use technologies that allow us to examine the function of the regulatory elements of a gene, those elements that control the rate at which RNA polymerase binds to the DNA and initiates transcription.
allow us to begin to understand the regulatory mechanisms that control gene expression.
We can use these different rates of evolution to help identify putative regulatory region(s) for a gene by
comparing genome sequences between two species that are much more closely related
Reporter gene technology allows us to
examine gene expression within a cellular context, in a simple and reliable fashion.
study cell metabolism within a cellular context, in a simple and reliable fashion.
detect mutations within a cellular context, in a simple and reliable fashion.
analyze protein structure within a cellular context, in a simple and reliable fashion.
Reporter gene studies are very important in
the initial analysis of a promoter
the transcription factors that may bind to it
the construction of a transgenic organism.
The most direct, precise and reliable way of locating a promoter, or any other DNA sequence that influences transcription through the binding of specific proteins (which includes many but not all regulatory elements), is to detect
the binding of those proteins to specific DNA fragments.
the binding of those proteins to specific RNA fragments.
Four methods to detect the binding of those proteins to specific DNA fragments :
Northern Blotting, Chromatin Immunoprecipitation (ChIP), DNase Footprinting, and Western Blotting
Yeast one-hybrid assays, Chromatin Immunoprecipitation (ChIP), DNase Footprinting, and Western Blotting
Yeast one-hybrid assays, Chromatin Immunoprecipitation (ChIP), DNase Footprinting, and gel retardation assays
