Term
-in lab 7, we prepared squashes of __ chromosomes from __
--As you recall from Chapter 14 of the text, the typical cell division cycle consists of a single DNA replication cycle (__ phase) to generate a pair of sister chromatids, which are then segregated into individual daughter nuclei during mitosis (__ phase).
-- there are __ chromosomes/linkage groups in the drosophila genome that combine to form what is known as the __ |
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Definition
-polytene; drosophilia salivary glands
-- (s phase); (m phase)
-- four; chromocenter |
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Term
- in lab 7, the __ was removed from the larvae
- the larvae were dissected in a drop of __ to keep them fresh, and the removed glands were placed in another drop of __ |
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Definition
-salivary glands
- saline; saline |
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Term
- a new slide was then prepared with __
-- before transferring the salivary glands to the stained slide above, the glands were placed in __ for 30 seconds before moving them to the slide with __ |
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Definition
- lacto/aceto orecin stain
-- 45% acetic acid; lacto/aceto orcein |
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Term
| Compare the scale bars for the photomicrographs of the polytene chromosomes in Figure 1 and the metaphase chromosomes from a mitotically active cell in Figure 2. What can you conclude about their relative sizes from this? |
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Definition
| The polytene chromosomes in salivary glands are MUCH larger. They appear to be both longer and wider. |
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Term
| How would you describe the differences in banding patterns for the two types of chromosomes? Is it easier to see the bands in one type than in the other? |
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Definition
| The bands are much more visible in the polytene chromosomes of salivary glands. |
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Term
| The polytene chromosomes are both longer and wider than the chromosomes in the larval brain squash. The atypical cell cycle of the salivary gland cell can account for them being wider than a metaphase chromosome? What might account for them also being longer? [Consider the effect of cell cycle on chromosome organization. (Figure 12.12)] |
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Definition
| The metaphase chromosomes have undergone the final level of condensation characteristic of M phase. The polytene cells lack an M phase, so will not undergo this condensation. |
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Term
| Figure 10 shows immunostaining of a polytene chromosome spread with an antibody against a chromosomal protein. How would you describe the immunolocalization pattern of this protein? Which region of the chromosome is most highly stained with the Texas Red-labeled antibody? |
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Definition
| It is staining the centromeric heterochromatin |
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Term
| . Based on this localization pattern and what you learned in Chapter 12 about heterochromatin structure, what protein do you think is recognized by the antibody |
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Definition
| HP1 (or Lysine 9-methylated histone H3) |
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Term
| Are there any other proteins (or covalently modified form of histone) you would expect to have a similar staining pattern? |
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Definition
| Lysine 9-methylated histone H3 (or HP1) |
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Term
-In lab 9 we will use __ as a means to monitor activity of the __ pathway in Drosophila larvae
-The __ pathway is a highly conserved pathway for sensing and responding to high __ levels in the blood stream or __ of multi-cellular animals
-__ produce glucagon in response to low glucose levels in the bloodstream; __ produce insulin in response to high levels |
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Definition
- gfp tagging; insulin signaling
- insulin signaling; glucose; hemolymph
- pancreatic alpha cells; beta cells |
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Term
-The Insulin Signaling Pathway. Insulin from the bloodstream binds the __, stimulating its __ and activation of __. __ (previous) phosphorylates __ in the plasma membrane allowing recruitment of __ to the membrane. These kinases then phosphorylate downstream targets to bring about __ and __.
--The final kinase to be activated in the pathway is __, which brings about the cellular response. __ stimulates the glucose transporter to become localized on the plasma membrane to allow glucose uptake from the bloodstream. |
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Definition
- insulin receptor; auto-phosphorylation; PI-3 kinase; PI-3 kinase; lipids; ph-domain kinases; glucose uptake; cell growth
- PKB |
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Term
-In lab 8, we will monitor the activity of the insulin signaling pathway in Drosophila through use a __ version of the __
-- the indicator protein is called __ |
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Definition
- gfp tagged; ph domain
-- tGPH |
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Term
| The GAL4/UAS binary system. __ protein expressed from the __ transgene drives tissue-specific expression of the activated __ from a __ binding site for __ protein |
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Definition
| - GAL4; en-GAL4; insulin receptor (InR); UAS; GAL4 |
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Term
| -We will use two different tissue-specific promoters to drive expression of GAL4 (and, thus, UAS-InR1325D) in today’s exercise. One will drive expression in the eye (__); the other (__) will drive expression in the back (posterior) half of each segment of the larval cuticle, as well as in organs located in the posterior half of the animal. In the larval cuticle, the cells in the neighboring anterior half of each segment can provide a negative control to those in the posterior half expressing __. |
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Definition
| (GMR-GAL4); (en-GAL4); (UAS-InR) |
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Term
| Why are the eyes of the animals expressing the activated InR in the eye larger than those of animals not expressing it? Examine the surface of the eyes in the two animals at high magnification. Does the increased eye size appear to be the result of an increase in the size of individual cells (cell growth) or an increase in the number of cells (cell division)? |
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Definition
| The insulin signaling pathway integrates information about nutritional status, ATP, levels and developmental cues to regulate cycle progression and cell growth, as well as glucose uptake. An activated insulin signaling pathway induces increased cell division and/or growth. You may be able to see that the eyes of animals expressing activated InR in the eye have an increased number of eye cells. This suggests that the insulin signaling pathway induced increased cell division, rather than cell growth, in the eye. |
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Term
| Is the subcellular distribution of the tGPH different in the cells of the stomach, intestine, or fat body tissue dissected from the long bodied (expressing activated InR) vs. the short bodied larvae (not expressing activated InR)? Give an explanation for any difference you observe. |
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Definition
| You may be able to see faint membrane-specific localization of the tGPH reporter in the short bodied larvae, reflecting the active insulin signaling in those tissues of wild type larvae. This signal becomes much brighter and more distinct in the long bodied larvae that are expressing the activated form of the InR, probably due to concentration of the GFP signal in the membrane. (It is unlikely that the enhanced signal for the tGPH involves induction of transcription of the tGPH reporter by InR1325D, as the promoter for the reporter is not insulin-regulated.) |
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Term
| Is the subcellular distribution of the tGPH uniform throughout the empty larval cuticle case, or are you able to discern a pattern of GFP expression in the cuticle case? Give an explanation for any pattern you observe. |
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Definition
| You may be able to see a striped pattern of GFP expression in the cuticle case. This reflects the concentration of the tGPH in the plasma membrane of cells in the posterior half of each segment. The PH domain of the tGPH reporter becomes localized on the plasma membrane in response to activation of the insulin signaling pathway. The InR1325D mutation constitutively activates this pathway in the cells where it is expressed (posterior cells of each segment). tGPH is an indicator of an activated insulin signaling pathway. tGPH does not concentrate in the plasma membrane in the anterior half of each segment where InR1325D is not expressed. This gives rise to the striped pattern. |
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Term
| . Were the cells larger or smaller in the back half of each segment, where activated InR was expressed? How do you explain this? (It may be easier to see this in a filet of the larval cuticle case. |
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Definition
| The insulin signaling pathway integrates information about nutritional status, ATP, levels and developmental cues to regulate cycle progression and cell growth, as well as glucose uptake. An activated insulin signaling pathway induces increased cell division and/or growth. Judging from the increased size of the cells expressing activated InR, insulin signaling appears to induce increased cell growth, rather than cell division, in the cells lining the larval cuticle. |
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Term
| How do you explain the effect of the InR1325D mutation on the activity of the Insulin Receptor? Assuming that Tyrosine is present at amino acid 1325 in the wild type InR, why does this mutation cause the receptor to be constantly (constitutively) active? |
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Definition
| This is a mutation apparently replaces the phosphorylatable Tyrosine residue of this Receptor Tyrosine Kinase with an Aspartic Acid, mimicking the effects of phosphorylation of this residue. This mutant protein will constantly activate the downstream targets of the insulin signaling pathway. |
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Term
| Mutation of another amino acid in the transmembrane domain of the InR to Aspartic Acid also results in a constitutively activated insulin signaling. How might you explain this effect? |
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Definition
| This suggests a role for the transmembrane domain in insulin signaling. Insulin binding to the receptor might trigger a conformational change in the transmembrane domain, similar to that associated with the mutation, that is in some way needed for its activation. For example, it may stimulate the receptor to form a dimer with a second activated InR or may allow it to interact with one of its downstream target proteins on the cytoplasmic face of the membrane |
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Term
| How might the cell cycle regulation of the epidermal cells expressing the activated InR differ from those not expressing it to stimulate them to grow larger? (Think molecules....Consider what you learned in Ch 14 about cell cycle regulation.) |
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Definition
| The activated InR must stimulate the cells to remain in G1 and continue to grow in size before entering another S phase. They could do this by preventing destruction of p27, needed for entry into S phase. Or the cells could, like salivary glands, become polytene by entering multiple S phases, without intervening M phases. Entry into M phase could be inhibited by preventing synthesis of M phase-promoting cyclin (cyclin B) at the end of each S phase. |
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Term
| How might the cell cycle regulation of the eye cells expressing the activated InR differ from those not expressing it to cause them to continue dividing? (Think molecules....Consider what you learned in Ch 14 about cell cycle regulation.) |
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Definition
| The activated InR must stimulate the cells to enter S phase more readily (by stimulating destruction of p27 or production of S phase-promoting cyclins (E and A)). |
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