You are studying the mushroom gene ZIP. which you have cloned. ZIP is not transc
ID: 67059 • Letter: Y
Question
You are studying the mushroom gene ZIP. which you have cloned. ZIP is not transcribed in young mushrooms, but is transcnbed in old mushrooms. Previous studies had suggested that the two genes encoding HLH transcriptional factors GOO and DEE are required to express ZIP. Describe an assay that you could use to test whether the transcription of ZIP is regulated by changes in chromatin structure during mushroom aging. Assuming that the sequences of the genes encoding GOO and DEE are known, describe how you might use the yeast two-hybrid method to test if those two proteins physically interact. Include in your answer a description of components of the vectors and yeast strains that you will use.Explanation / Answer
2a.
Based on the given data, the ZIP gene regulation by chromatin structures can be identified as follows:
G banding assay can be used to identify the change in the chromosome. Consider in the given case, the position and location of gene on chromosome is known.
Isolate the genetic material from both young and old age mushrooms.
Stain the mixture with Giemsa stain.
Treat the material with trypsin that denatures the proteins.
Observe the stain intensity; the darkly stained chromosomes indicate heterochromatin state, while light stained chromosomes indicate euchromatin.
The genes cannot be expressed in heterochromatin state. In the given case, may be the genes are in heterochromatin state at young age and euchromatin state at old age.
2b.
Two-hybrid technique is used to discover protein-protein interactions and protein-DNA interactions. In this technique, the downstream reporter genes are activated by binding of transcription factors onto upstream activating sequence.
According to the given information, the genes encoding GOO and DEE are known. Thus, the DNA-binding domain and protein (BAIT) for both genes (GOO and DEE) are prepared. These are inserted into yeast cell along with ZIP gene. If the transcriptional factors GOO and DEE interact and bind to ZIP then the gene is expressed. Thus, expression of ZIP gene indicates the GOO and DEE are interacting with one another.
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