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

Question 260

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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.
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. <strong>Figure 1</strong>  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.<strong>Table 1</strong>  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. -A recent study of a model organism showed that HMG-5, another member of the HMG family, mainly localized to the end of chromosomes.  The site of HMG-5 localization would correspond to regions in humans that: A) contain multiple copies of different DNA sequences. B) contain heterochromatin similar to that of centromeres. C) are subsequently lengthened with each round of cell division. D) are actively replicated by the enzyme DNA polymerase. 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
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. <strong>Figure 1</strong>  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.<strong>Table 1</strong>  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. -A recent study of a model organism showed that HMG-5, another member of the HMG family, mainly localized to the end of chromosomes.  The site of HMG-5 localization would correspond to regions in humans that: A) contain multiple copies of different DNA sequences. B) contain heterochromatin similar to that of centromeres. C) are subsequently lengthened with each round of cell division. D) are actively replicated by the enzyme DNA polymerase. 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.
-A recent study of a model organism showed that HMG-5, another member of the HMG family, mainly localized to the end of chromosomes.  The site of HMG-5 localization would correspond to regions in humans that:


A) contain multiple copies of different DNA sequences.
B) contain heterochromatin similar to that of centromeres.
C) are subsequently lengthened with each round of cell division.
D) are actively replicated by the enzyme DNA polymerase.

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