Self-assessment worksheet
Gene fusions, GUS, GFP and microscopy.
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Library/Internet exercise:
Answer the following questions:
1. Draw a diagram of a typical protein-coding plant gene. The annotation should include the following listed elements, and indicate their typical positions within the gene. Briefly describe the properties of the elements.
promoter
upstream regulatory sequences
enhancer
silencer
RNA polymerase initiation site
intron
exons
UTRs
START codon
STOP codon
polyadenylation site
2. What are transcription factors, and how do they interact with genes?
3. What is a reporter gene, and how is it detected?
4. Draw a schematic view of the differences between plant transformation vectors that might be used to produce protein fusions, transcriptional fusions and for enhancer detection in plants.
5. Why are these different gene fusions useful?
6. Both green fluorescent protein (GFP) and ß-glucuronidase (GUS) are widely used as reporter genes in plants. Describe major advantages of GFP over GUS, and vice versa.
7. As a keen plant biologist, you wish to construct your own fluorescent microscope for work with a variant of green fluorescent protein. The excitation and emission spectra of the protein are shown below. You have access to a box of filters that transmit or reflect light in the following bands:
Bandpass Filter 1: 350-460nm; Filter 2: 450-490nm; Filter 3: 515-560nm, Filter 4: 550-570nm
Beamsplitter Mirror 1: 460nm; Mirror 2: 500nm; Mirror 3: 580nm; Mirror 4: 595nm
Longpass Filter 1: >470nm; Filter 2: >530nm; Filter 3: >580nm; Filter 4: >635nm
You need to construct a suitable filter block for imaging GFP. What are the roles of the different types of filter in the microscope lightpath? What filters will you choose for the (i) excitation filter, (ii) beamsplitter and (iii) emission filter. Explain why you chose this combination?