Lab Final

Which experiments could be used to test your prediction from the DNA sequences expected to be present on the polytene chromosomes, but absent from mature mRNA?Chromosomes in situ hybridization with labeled DNA probe.Which proteins would you expect to be present on polytene chromosomes of larval salivary glands, but absent on metaphase chromosomes from larval brains?DNA polymeraseWhich experiments could be used to test which proteins would be present on polytene chromosomes of larval salivary glands, but absent on metaphase chromosomes from larval brains?Immunostaining the chromosomes with antibodies that recognize that protein Expression of a GFP-tagged version of that protein.In the diagram of the dissected Drosophila larva, which arrow is pointing to the salivary gland used in your polytene chromosome squashes?Arrow B Arrow pointing to the structure(s) along the outside of the "three balls" near the middle of the head of the larva.In the diagram of the dissected Drosophila larva, which arrow is pointing to the fat body tissue?Arrow C Arrow pointing to the structure along the length of the body stretching from below the salivary glands down to at least half the length of the body of the larva.In the enlarged region of a polytene chromosome, which best describes how the chromatin in the light band indicated by arrow A compares to that in the dark band indicated by arrow B?The chromatin in the light band is less compact than the chromatin in the dark band, and are competent for transcription.Describe the enlarged light band (puff) indicated by arrow C in comparison to the narrower light band indicated by arrow A.The DNA in the puff is being transcribed at a higher level than the DNA in the narrower light band.Chromosome squashes can be used to detect mutations in genes. Which mutations would be most likely to be detected by this method?Transposable Element insertion into it.In Lab 4B you used a Drosophila stock expressing a GFP-tagged PH domain as a reporter for an activated insulin signaling pathway. The transgene for the GFP-tagged PH domain provides a good reporter for activation of this pathway because _________.the PH domain targets proteins to the plasma membrane during pathway activation.Which tissues would membrane localization of the tGPH reporter be observed in the absence of the constitutively active mutant form of InR in well-fed larvae?Fat body tissueThe eyes of the animals expressing a constitutively active mutant form of the Insulin Receptor (InR) from the eye-specific GMR promoter were __________.Larger than the eyes of wild type animals, because the cells were stimulated to undergo extra divisions.The engrailed GAL4 driver (enGAL4) was used to drive expression of the consitutively active mutant form of InR (InR^Y13250) in which tissues?Epithelial cells lining the cuticle case in the posterior (back) region of each segment.Under which conditions would the tGPH reporter of Lab 4B not be membrane-localized?In starved larvae In larvae carrying a loss of function mutation in PI3 kinase In larvae carrying a loss of function mutation in InRIf a PH-GFP fusion protein were expressed in a human cell line derived from a patient with Diabetes Type II, which sub-cellular locations would you expect to find it?CytoplasmIf a GFP-tagged GLUT4 transporter were expressed in a human cell line derived from a patient with Diabetes Type II, in which of the following sub-cellular locations would you expect to find it?Secretory vesicles in cytosolIn the GAL/UAS binary system, __________ acts as a transcription factor that binds and activates transcription from the __________ sequence located at the 5' end of a transgene.GAL4, UASIn a cell expressing a constitutively active PI3 kinase along with an inactive Akt (present but non-function kinase activity) of the survival/growth factor response pathway, would you expect the pathway to be constitutively activated or unresponsive?It will be inactive, because Akt acts after PI3 kinase. Even though PI3 kinase is constitutively active it can have no effect on the inactive Akt.How would you expect each kinase (constitutively active PI3 and inactive Akt) to be localized in the cell (cytosolic or membrane-associated)?Because PI3 is constitutively active, IP# on membranes will be phosphorylated, allowing Akt to be membrane-associated. PI3 kinase will be associated with membranes while phosphorylating the lipids, but will also be cytosolic.In a cell expressing constitutively active Akt with an inactive PI3 kinase (present but non-functioning kinase activity), would you expect the pathway to be constitutively activated or unresponsive?It will be constitutively active, because Akt acts after PI3 kinase. Even though PI3 kinase is inactive constitutively active Akt doesn't need it.How would you expect the constitutively active Akt and inactive PI3 kinase to be localized in the cell (cytosolic or membrane associated)?Because PI3 kinase is inactive IP3 on membranes will be unphosphorylated, preventing Akt from being membrane-associated. PI3 kinase will not be associated with membranes.Imagine that your are given a cell line that was derived from a human diabetes patient. A transgene for a GFP-tagged GLUT 4 transporter does not become membrane localized in the cell in response to the addition of insulin to the culture medium. Design experiments to exhaustively analyze the abundance, size, and phosphorylation status of all known protein components of the pathway to determine if the mutation lies in a known component. You can use any of the techniques you learned about in the course. You also have access to antibodies for each of the the known protein components, including those that have specificity for particular phosphorylated forms of the proteins.Use Northern blotting and Western blotting on all known protein components of the pathway, and transcripts for them, to determine the the abundance or size of one of these proteins is altered in the diabetes cell line. PCR can be used to look for large alterations in sizes of DNA fragments of the genes for each, and RT-PCR is an alternative method that could be used to monitor changes in transcript abundance and size. The phospho-specific antibodies could be used to determine if InR, PI3K, and Akt become appropriately phosphorylated in response to insulin.Imagine that your are given a cell line that was derived from a human diabetes patient. A transgene for a GFP-tagged GLUT 4 transporter does not become membrane localized in the cell in response to the addition of insulin to the culture medium. In the event that your experiments failed to find a mutation in a known component of the pathway, design additional experiments that could determine the point in the pathway where the mutated gene must function. You might want to use an approach based on the double mutant phenotypes, and/or make use of GFP-reporter transgenes similar to the one you used in Lab4B (GFP-tagged PH domain). You are able to introduce transgenes into the cell lines to accomplish this, and the antibodies with specificity for particular phosphorylated forms of the proteins are available.The fact that the cell line was derived from a diabetes Type II patient and the failure of the GFP-tagged PH domain to become membrane-localized in it suggest that cell line carries a loss-of function mutation in the pathway. To determine the point in the pathway where the mutated gene functions, transgenes for constitutively activated forms of InR, PI3K, and Akt can be introduced into the cells. If the loss of function mutation is in a gene for a protein that acts after InR, addition of constitutively activated InR will not permit membrane localization of GFP-PH in the presence of insulin. If the mutation is in a gene that acts before InR, it would then allow GFP-PH membrane localization even in the absence of insulin. The same applies to introduction of constitutively active PI3K or Akt. The combination of experiments with constitutively active forms of each protein added one at a time could define the exact point in the pathway at which the mutation lies. Similarly, the phosphor-specific antibodies can be used to determine if there is a defect in phosphorylation of InR, PI3K, and Akt. If all three are phosphorylated in the presence of insulin, it is assumed that the mutated gene must function after Akt in the pathway. But if, for example, PI3K is appropriately phosphorylated (in the presence of insulin) but Akt is not, the mutated gene must function between the PI3K and Akt steps.Imagine that you are given a cell line that was derived from a human liver cancer patient. A transgene for a GFP-tagged GLUT4 transporter remains membrane localized in the cell, even in the absence of insulin added to the culture medium. Design experiments to exhaustively analyze the abundance, size, and phosphorylation status of all known protein components of the pathway to determine if the mutation lies in a known component. You can use any of the techniques you learned about in the course to do this. And assume that money is no obstacle. You have access to antibodies for each of the know protein components, including those that have specificity for particular phosphorylated forms of the proteins.Use Northern blotting and Western blotting on all known protein components of the pathway (and transcripts for them) to determine if the abundance or size of one of these proteins is altered in the diabetes cell line. PCR can be used to look for large alterations in sizes of DNA fragments of the genes for each, and RT-PCR is an alternative method that could be used to monitor changes in transcript abundance and size. The phospho-specific antibodies could be used to determine if InR, PI3K, and Akt become inappropriately phosphorylated even in the absence of insulin.Imagine that you are given a cell line that was derived from a human liver cancer patient. A transgene for a GFP-tagged GLUT4 transporter remains membrane localized in the cell, even in the absence of insulin added to the culture medium. In the event that your experiments above failed to find a mutation in a known component of the pathway, design additional experiments that could determine the point in the pathway where the mutated gene must function. You might want to use an approach based on the double mutant phenotypes, and/or make use of GFP-reporter transgenes similar to the one you used in Lab4B (GFP-tagged PH domain). And be aware that you are able to introduce transgenes into the cell lines to accomplish this.The fact that the cell line was derived from a human liver cancer patient and the observation that GFP-tagged PH domain is membrane-localized even in the absence of insulin added to the culture medium suggest that cell line carries a gain-of function mutation in the pathway. To determine the point in the pathway where the mutated gene functions, transgenes for RNAi directed to the genes for InR, PI3K, and Akt can be introduced into the cells. If the gain of function mutation is in a gene for a protein that acts after InR, addition of RNAi for InR will abolish the membrane localization of GFP-PH in the absence of insulin. If the mutation is in a gene that acts before InR, it would continue to allow GFP-PH membrane localization even in the absence of insulin. The same applies to introduction of constitutively active PI3K or Akt. The combination of experiments using RNAi directed against the gene for each protein individually could define the exact point in the pathway at which the mutation lies. Similarly, the phosphor-specific antibodies can be used to determine if there is constitutive phosphorylation of InR, PI3K, and Akt in the absence of insulin. If all three are phosphorylated in the absence of insulin, it is assumed that the mutated gene must function at some point before Akt in the pathway. But if, for example, PI3K is not phosphorylated in the absence of insulin but Akt is, the mutated gene must function between the PI3K and Akt steps.