You have isolated total RNA from muscle cells and constructed a muscle cDNA libr
ID: 77819 • Letter: Y
Question
You have isolated total RNA from muscle cells and constructed a muscle cDNA library. You wish to study the regulatory region of muscle-specific cDNA gene (gene M) that you have previously identified.
(a) For you study, you need to isolate a genomic clone of gene M. Why is a cDNA clone of gene M not appropriate for your study?
(b) Outline the steps you would take to isolate the genomic gene M
(c) You have isolated a genomic clone for gene M which contains the entire coding region as well as 1.2kb of the upstream regulatory (promoter) region. A map of genomic gene M is shown below. You have also prepared a reporter lacZ fusion gene constructed by cloning a 1.3kb BamH1 upstream fragment of gene M into a plasmid vector containing the lacZ gene. This fusion gene, when injected into cultured muscle cells, is capable of expressing -galactosidase activiy. What experiment would you carry out next to determine if the fusion construct is developmentally regulated?
(d) Using an antibody that is specific for the -galactosidase protein, you analyzed the tissues of a "gene M-lacZ" transgenic mouse and found that "protein M" is also expressed in bone cells of the mouse. How would you determine whether the same transcript is expressed in muscle and bone cells? If different size mRNA transcripts were obtained, suggest some possible reasons for the observation.
(e) You decided to analyze the promoter region of gene M to identify regulatory DNA elements that control gene M expression in muscle and bone cells. The results obtained with the lacZ fusion constructs containing different fragments of the 5' promoter region of gene M are shown below. Which DNA region (s) are important for specifc expression in muscle? and in bone? Explain.
BamHI BamHI bp 300 400 50 350 100 it Coding region S S S Sau3A Expression Expression DNA in subclone in muscle in boneExplanation / Answer
Answer for (a): A cDNA clone of gene M is not appropriate for the study because the cDNA clone does not include any introns or regulatory regions that are responsible for controlling the expression of the gene. The regulatory regions are responsible for encoding enhancers or promoters that are needed for the expression of the gene. For example, if a gene is expressed highly in liver and lowly expressed in heart, these regulatory regions control the expression level. In the absence of the regulatory regions either the gene will be having homologous expression in liver and heart or no expression level.
Answer for (c) : I would create a transgenic mouse. The transgenic mouse was created by injecting the linearized plasmid carrying the fused Gene M – LacZ into mouse pro-nuclei. After injection the pronuclei will be placed in pseudo pregnant mothers. Embryos will be formed in these mice and the pregnant mothers will be sacked at different time points of embryonic stages such as E10.5, E12.5, E14.5, E16.5, E18.5 and P0 (Pups after birth). Perform whole mount staining for beta-galactosidase activity on these embryos. Check the staining patterns and see if lac Z staining increases from E10.5 to P0. This confirms whether the gene is developmentally regulated or not.
Answer for (d): To check if the same transcript is expressed in both muscle and bone cells, I would perform an RNA-Seq analysis. I would isolate mRNA from both muscle and bone separately. After isolating the RNA samples, cDNAs will be synthesized from the pool of mRNAs. Perform sequencing analysis and check if cDNAs of gene M match in mRNA isolated from muscle and bone. If they match, it can be concluded that no alternative transcripts are formed. If not then the transcripts between bone and muscle differ.
A second way of determination is by RT-PCR. Check the sequence of gene M transcripts from any of the databases like ensembl, mgi etc. Design primers for each and every transcript. Verify that at least one of the primers (either forward or reverse) is variable to all the transcripts. Note down the predicted PCR product sizes for each set of primers. Perform PCR reaction on mRNA from bone and muscle with all the given sets of primers. Run the PCR reactions on agarose gel and compare the product profiles between muscle and bone. If the product profiles are dissimilar then it can be concluded that different transcripts are expressed in bone and muscle.
Possible reasons for obtaining different mRNA transcripts:
1. Alternative splicing.
2. Regulatory elements present in bone and muscle.
Answer for (e): The promoter region that is responsible for expression of gene M in bone is as follows from the given map:
On the other hand the expression of gene M is regulated by the following sequences according to the map:
From this data, it can be clearly seen the DNA sequences present in 2, 3 and a, b are responsible for expression of M in bone and muscle. But this was not observed in DNA sequences 1 and c. The presence of these sequences is exclusively responsible for expression in bone and muscle. Hence the answer is: The regulatory elements responsible for gene M expression in bone is the 750bp DNA sequence present between BamHI and the third Sau3A site. On the other hand the regulatory elements responsible for gene M expression in muscle are the 450bp between the third Sau3A site and the second BamHI site.
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