7.012 Problem Set 5. Question 1

Transcription

1 Name Section Problem Set 5 Question 1 While studying the problem of infertility, you attempt to isolate a hypothetical rabbit gene that accounts for the prolific reproduction of rabbits. After much effort, you finally map a gene on the Y chromosome in rabbits that may account for the high fertility of rabbits. You name this gene High Reproductive Gene Y-Chromosome, or hrgy for short. You want to test the effect of expressing the rabbit hrgy gene in a mouse. You inject the linear DNA fragment for the rabbit hrgy gene into fertilized mouse eggs at the single cell stage. This DNA randomly inserts into the mouse genome. The mice that result from successful injections have the hrgy gene integrated at the same position in the genome in every cell. This mouse is referred to as a transgenic mouse. a) Which of the following 2 fragments of rabbit genomic DNA would you pick to inject into the mouse and why? promoter rabbit genomic DNA ATG TAG hrgy gene Fragment 1 Fragment 2

2 Question 1 cont. You ve successfully injected the 2200 bp rabbit hrgy DNA into the single-cell fertilized mouse eggs. From the eggs you derive baby mice. To confirm that these mice carry the rabbit hrgy gene, you perform PCR. Your PCR reactions have a sample of genomic DNA from each of your baby mice as template. Using the appropriate primers, you amplify a 380 bp fragment within an intron of the rabbit hrgy gene. The DNA amplified is subjected to gel electrophoresis. b) In lane 1 of the gel below, draw the DNA band(s), if any, that you would see, if the DNA was amplified from a mouse carrying the rabbit hrgy gene. In lane 2 of the gel, draw the band(s), if any, you would see in a mouse that doesn t carry the rabbit gene. MW Markers Lane 1 Lane c) After performing the PCR reactions, you find a mouse carrying the rabbit hrgy DNA. Is this sufficient to tell you that the gene is expressed? If not, what other molecules should you look for and why? d) You check for hrgy expression in many different tissue types of the transgenic mouse, and find that it is only expressed in one organ in the mouse - the testis. However, you know that the hrgy gene is present in every cell. What might be found in testes cells that could account for this very tissue specific expression of the hrgy gene? 2

3 Question 2 Using the known DNA sequence of hrgy, you derive the amino acid sequence of the HrgY protein to get clues as to its function. By considering the polarity, charge, and positional order of the amino acids in HrgY, you notice that it contains a transmembrane domain and sites for glycosylation. a) i) What are the key features of a transmembrane domain? Include the kind of amino acids that would be found in a transmembrane domain. ii) Where would you find proteins with transmembrane domains? iii) Name 2 ways that membrane proteins function in a cell. iv) What is glycosylation? v) In which organelle does glycosylation take place? b) If the Signal Recognition Particle could be absent in a cell, would you expect. Circle one 1 transmembrane proteins in that cell to be glycosylated? YES NO 2 transmembrane proteins to be fully translated? YES NO 3 the genes encoding the transmembrane proteins to be transcribed? YES NO 4 signal sequences to be cleaved? YES NO 5 secreted proteins to be exported? YES NO 3

4 Question 3 By comparing the HrgY amino acid sequence with other known proteins, you observe that the HrgY protein shares astounding homology with other tyrosine kinases receptors. a) What is the function of a tyrosine kinase (TK) domain? b) In order to determine whether the TK domain has significance for the HrgY protein, you create a mutant version of the hrgy gene that encodes a constitutively active tyrosine kinase domain within the HrgY protein. This mutant protein is no longer accessible to phosphatases. Would the mutant protein be found in a phosphorylated or nonphosphorylated state (Circle one). To ascertain what effect the constitutively active HrgY protein might have, you clone the mutated hrgy gene into a mammalian expression vector (a plasmid that replicates in mammalian cells). You transfect (similar to transformation in bacteria) normal mouse testes cells in tissue culture with your vector constructs. You compare your transfected mouse cells that contain the vector carrying the mutated hrgy to a group of control mouse cells that were transfected with just the vector or the vector carrying the wild-type hrgy gene (see below). You observe that the mouse cells synthesizing the mutant HrgY protein, show an strikingly large increase in the levels of another protein, which you name Protein X. Normal mouse testes cells Transfect with vector alone. Transfect with vector carrying wildtype hrgygene. Transfect with vector carrying mutant hrgy gene encoding constitutively active TK domain in the HrgY protein. Measure protein production Measure protein production Measure protein production low levels of Protein X low levels of Protein X high levels of a Protein X c) Explain, in general terms, how a constitutively active tyrosine kinase in the plasma membrane could cause the increase of Protein X within the cell. Be sure to include transcription in your answer. 4

5 Question 4 You finally determine that the introduced hrgy gene has no effect on the mouse s fertility. Although you learned little about hrgy from your transgenic mouse line, you decide to keep studying it, because at this point you have already invested a year of research into this gene and you are loath to start a completely new project. In a long shot experiment, you grow cells, that you isolated from the testes of a transgenic mouse carrying the wildtype rabbit hrgy gene, in medium, and measure Protein X synthesis. In these cells Protein X production is very, very low. However, when you grow these cells in medium supplemented with serum from rabbits, you find that the expression level of Protein X increases dramatically. (See picture below.) Note: Normal mouse testis cells grown in rabbit serum show no increase in Protein X levels (data not shown). Isolate testes cells from transgenic mouse expressing rabbit hrgy gene. mouse testes cells Split into 2 petri dishes. Grow in the presence of rabbit serum Grow without rabbit serum Measure Protein X production High Protein X production Low Protein X production a) How might you explain this? b) You have previously determined that the hrgy gene is being expressed in the testes of the male mouse. Given the experiment described above, what is one reason it may not alter the mouse s fertility? 5

6 Question 4 cont. After further study (you are now in your fourth year of UROP), you successfully purify a component from the rabbit serum that was able to induce Protein X expression in the transgenic cells. You determine that the component is a secreted protein. You name this secreted serum protein, SecC. c) Would the SecC protein purified from the serum have an attached signal sequence? Why? d) Would you expect the SecC protein to have a signal sequence at any point in its synthesis? If yes, when? Curious as to the effect of the SecC protein in living transgenic hrgy mice, you insert the gene encoding SecC into the genome of a virus. You infect the transgenic mice with this virus that will integrate its DNA (including the secc gene) into the genome of any mouse cells that it infects. Interestingly, your transgenic mice infected with the virus carrying the secc gene are demonstrating increased fertility. e) Would the virus carrying the secc gene need to infect the cells of the testes of the transgenic mouse to cause this increase in fertility? Explain your answer. 6