Analysis of Gene Expression

Two points concerning the measurements of mRNA levels:

• 1. 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. 2. the amount of mRNA present does not necessarily correlate with the amount of protein made.

• 1. 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. 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 method to measure protein levels in a sample
• a process to visualize cell structures under a microscope
• 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

• Identifies cell morphology
• Detects protein expression levels
• Measures cell viability

As an alternative to radioactive labelling, the probe in In situ hybridization can be labelled by:

• Direct visualization without labeling
• attaching a fluorescent dye

• Labeling with radioactive isotopes
• Enzymes like alkaline phosphatase or horseradish peroxidase

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.

• Promoter inhibition assays
• Promoter translocation studies
• 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

• Measuring the weight of different regions of a gene
• comparing genome sequences between two species that are much more closely related

• Comparing the color of different regions of a gene
• Analyzing the taste of different regions of a gene

Tags

NGSS.HS-LS4-2

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.

• Analyzing weather patterns

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.

• Western blotting
• Gel electrophoresis