Passage
Quantitative fluorescence recovery after photobleaching (FRAP) is used to study the movement of molecules in live cells. During FRAP, the fluorescence of a green fluorescent protein (GFP) -tagged molecule is first measured and then photodestroyed in targeted cell regions. Researchers then assess the time course for fluorescence recovery, which is an indicator of molecular mobility of the GFP-tagged protein into the photodestroyed region. The average time required to recover 80% of the pre-bleach fluorescence of protein histone 1 (H1) fused to GFP (H1-GFP) in the photodestroyed region is given as t80.FRAP was used to analyze the mobility of the architectural H1 alone and in the presence of high-mobility group (HMG) proteins. HMG proteins are dynamic modifiers that have been shown to have opposite effects but similar binding sites on chromatin structure.
Figure 1 Unique chromatin binding motifs of HMG proteinsDuring analysis, HMGA1 and HMGB1 were microinjected into the cytoplasm of mouse embryonic fibroblast cells expressing H1-GFP. FRAP was performed on euchromatin and heterochromatin. Euchromatin and heterochromatin domains were identified as regions weakly and strongly stained by H1-GFP, respectively. The relative intensity of H1-GFP fluorescence in euchromatin and heterochromatin of uninjected and injected cells was assessed, and t80 results are shown in Table 1.Table 1 Effect of HMGB1 and HMGA1 on H1-GFP Mobility
Adapted from Catez F, Yang H, Tracey KJ, Reeves R, Misteli T, Bustin M. Network of dynamic interactions between histone H1 and high-mobility-group proteins in chromatin. Mol Cell Biol. 2004;24(10) :4321-8.
-The experimental design described in the passage allowed researchers to do which of the following?Assess whether structural chromatin organization influences H1 mobilityAssess H1 and GFP competition at different levels of DNA compactionEvaluate if HMG proteins compete with H1 for chromatin-binding sites

Question

Passage
Quantitative fluorescence recovery after photobleaching (FRAP) is used to study the movement of molecules in live cells. During FRAP, the fluorescence of a green fluorescent protein (GFP) -tagged molecule is first measured and then photodestroyed in targeted cell regions. Researchers then assess the time course for fluorescence recovery, which is an indicator of molecular mobility of the GFP-tagged protein into the photodestroyed region. The average time required to recover 80% of the pre-bleach fluorescence of protein histone 1 (H1) fused to GFP (H1-GFP) in the photodestroyed region is given as t80.FRAP was used to analyze the mobility of the architectural H1 alone and in the presence of high-mobility group (HMG) proteins. HMG proteins are dynamic modifiers that have been shown to have opposite effects but similar binding sites on chromatin structure.

Figure 1 Unique chromatin binding motifs of HMG proteinsDuring analysis, HMGA1 and HMGB1 were microinjected into the cytoplasm of mouse embryonic fibroblast cells expressing H1-GFP. FRAP was performed on euchromatin and heterochromatin. Euchromatin and heterochromatin domains were identified as regions weakly and strongly stained by H1-GFP, respectively. The relative intensity of H1-GFP fluorescence in euchromatin and heterochromatin of uninjected and injected cells was assessed, and t80 results are shown in Table 1.Table 1 Effect of HMGB1 and HMGA1 on H1-GFP Mobility

Adapted from Catez F, Yang H, Tracey KJ, Reeves R, Misteli T, Bustin M. Network of dynamic interactions between histone H1 and high-mobility-group proteins in chromatin. Mol Cell Biol. 2004;24(10) :4321-8.
-The experimental design described in the passage allowed researchers to do which of the following?Assess whether structural chromatin organization influences H1 mobilityAssess H1 and GFP competition at different levels of DNA compactionEvaluate if HMG proteins compete with H1 for chromatin-binding sites

Quizplus · Accepted Answer

11ecba2d_0f3b_45e9_a3bf_f75854e495b7_MD0008_00 The researchers were interested in studying the molecular mobility of H1 in the two known forms of chromatin, heterochromatin and euchromatin, which differ greatly in compaction level. Based on this fact, it can be postulated that their first goal was to assess how the mobility of H1 is influenced by chromatin compaction and organization (Number I). The passage states that H1 and HMG proteins have similar binding sites on chromatin and therefore compete for the same regions (ie, if one binds, the other cannot). H1 mobility was studied alone and then in the presence of HMG proteins to determine if these proteins competed with H1 to bind chromatin at the same sites. Chromatin and euchromatin were studied to ensure that the two possible compaction levels were accounted for during the dynamic competition studies (Number III). (Number II) An objective of this experiment is to study the mobility of H1, not GFP. The GFP molecule serves only as the fluorescent tag that allows tracking of H1 movement within the cell, there is no competition between the two molecules. Educational objective: Chromatin compaction can influence accessibility of regulatory factors to DNA. The known H1 function is to stabilize chromatin compaction by securing nucleosome packaging. However, competing proteins would have the opposite effect, which would be to destabilize chromatin compactness and enhance accessibility to DNA.

Passage Quantitative Fluorescence Recovery After Photobleaching (FRAP) Is Used to Study