Figure 1. The DNA encoding ONLY the luminal domain of US3 was fused in-frame to

Question

Figure 1. The DNA encoding ONLY the luminal domain of US3 was fused in-frame to the DNA that codes for GFP (green fluorescent protein). When translated in the cell this fragment, called A128GFP, localized to the ER (Figure 1, panel B below). To determine which amino acids in the luminal domain carry the ER retention/ retrieval sequence, the US3 sequence in each fusion construct was shortened at the 3'-end to produce fusion proteins that contain C-terminal sequence from US3. Constructs are named for the amino acids they contain. For instance, ?128GFP contains amino acids 1-128 of US3 and A84GFP contains amino acids 1-84. The localization of each of these truncated fusion proteins was examined in cells. 5. Is this a find it, lose it and/or move it experiment? Explain your choice. US3 GFp 455GFP 4500FP FIG. 1. Essential domain for ER retention of the US3 glycoprotcis. (A) Schomatic diagram of the chimeric coestructs with the GFP reporter and US3 luwminal domain used to identify the reteation elements TM, transmemberane domainc CT,cysoplasmic tail. (1B) HeTa cells expressing the indicated miutants were analyned for intracellelar localization by immunotluonesoence microscopy. Similar results were obtained in at least three independent experiments 6. (1pt) Fig. 1 - Which US3-GFP (1pt) Fig. 1-What US3 amino acid sequences contain the ER retention/retrieval sequences? a. aa. 129-161 b. aa. 85-128 c. aa. 55-65 d. aa. 51-55 e. aa. 39-50 f. aa. 191-205 7. fusion constructs do NOT localize to the ER? Choose any that apply a. ?84GFP b. ?65GFP c. ?70GFP d. ?55GFP e. A38GFP

Explanation / Answer

In this experiment, luminal domain of US3 was added with a DNA which codes GFP. The idea behibd the eyperiment is to find the amino acid sequence which is essential for ER retention or localization. Different part of US3 were fused with GFP and then co-localization pattern were analyzed. So basically it is a find it experiment as the aim was to find the amino acid sequnce essential for ER localization.

6.The US3-GFP construct d) delta 55-GFP and e) delta-38GFP do not localize in the ER.

7. The construct-a-129-161

                            b-85-128

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