The platypus is an egg-laying mammal. Its eggs hatch 10 days after being laid, a
ID: 67176 • Letter: T
Question
The platypus is an egg-laying mammal. Its eggs hatch 10 days after being laid, after developing in utero for 28 days. One of its genes, Cbl1, encodes cystathionine-beta-lyase, which is used to make methionine.
5a. Describe a method that you could use to see if the level of Cbl1 mRNA in eggs changes between 0, 5, and 10 days after being laid.
5b. How could you determine if any change is due to altered activity of the gene's promoter (more initiation events), or altered rates of degradation of the mRNA in the older eggs?
5c. What controls would you need for these experiments?
Explanation / Answer
5a. Describe a method that you could use to see if the level of Cbl1 mRNA in eggs changes between 0, 5, and 10 days after being laid.
A number of methods are available to detect and determine the presence of mRNA in a cell.
Method
Application
Northern blot analysis
Provides information about transcript size
Nuclease protection assays (NPA)
Simultaneously examine multiple messages
In situ hybridization
Localize expression of a particular gene within a tissue or cell type
Reverse transcription-polymerase chain reaction (RT-PCR). Nested and quantitative real time
Detects and quantifies gene expression.
Northern analysis:-
One of the standard method to detect and quantify the mRNA levels.
5b. How could you determine if any change is due to altered activity of the gene's promoter (more initiation events), or altered rates of degradation of the mRNA in the older eggs?
The presence of altered rates of degradation of the mRNA in the older eggs can be determined using nuclear run on assays. This assay will measure the transcriptional activity of selected endogenous genes.
1) Collect the samples containing different steady state amounts of the mRNA. 2)The cells are chilled and membranes are permealized or lysed and this will cause polymerase pausing.
3) Incubate the nuclei at 37C in the present NTPs and radiolabeled UTP. No new transcripts will be synthesized.
4) But the radiolabeled nucleotide will be incorporated into the transcripts when the cells were first chilled and lysed.
5) The number of nascent transcripts on the gene at the time of chilling is proportional to the frequency of transcription initiation. Now determine the number of nascent transcripts from the amount of radioactivity hybridizes to the membrane.
Like this we can measure the rates of mRNA degradation in different types of cells.
5c. What controls would you need for these experiments?
For the experiment 5a, we can keeps Cbl1 mRNA in eggs on day 0 as control, so that we can compare mRNA levels on 5th and 10th day with it.
For the experiment 5b, we can take younger eggs as control, so that the rate of mRNA degradation in older eggs can be compared with them.
Method
Application
Northern blot analysis
Provides information about transcript size
Nuclease protection assays (NPA)
Simultaneously examine multiple messages
In situ hybridization
Localize expression of a particular gene within a tissue or cell type
Reverse transcription-polymerase chain reaction (RT-PCR). Nested and quantitative real time
Detects and quantifies gene expression.
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