Limits...
Correlations of three-dimensional motion of chromosomal loci in yeast revealed by the double-helix point spread function microscope.

Backlund MP, Joyner R, Weis K, Moerner WE - Mol. Biol. Cell (2014)

Bottom Line: As controls, we tracked pairs of loci along the same chromosome at various separations, as well as transcriptionally orthogonal genes on different chromosomes.This relative increase has potentially important biological implications, as it might suggest coupling via shared silencing factors or association with decoupled machinery upon activation.We also found that on the time scale studied (∼0.1-30 s), the loci moved with significantly higher subdiffusive mean square displacement exponents than previously reported, which has implications for the application of polymer theory to chromatin motion in eukaryotes.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Stanford University, Stanford, CA 94305.

Show MeSH

Related in: MedlinePlus

Labeling scheme for each condition imaged. Green star corresponds to LacO/LacI-GFP label, and red star corresponds to TetO/TetR-mCherry label. TetO repeats were integrated at the same position relative to the GAL locus (<3 kbp from GAL1) wherever present. LacO repeats were integrated in the same position (i.e., <3 kbp from GAL1) for the DLDC and GFP cross-talk experiments. LacO repeats are immediately adjacent to the 5′ end of PES4 for the GAL/PES4 experiment. The DLDC and GAL/RPL9A cases were imaged in galactose and dextrose, whereas all other (control) cases were imaged in dextrose. Distances between loci and labels in schematic are not necessarily to scale.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4230621&req=5

Figure 2: Labeling scheme for each condition imaged. Green star corresponds to LacO/LacI-GFP label, and red star corresponds to TetO/TetR-mCherry label. TetO repeats were integrated at the same position relative to the GAL locus (<3 kbp from GAL1) wherever present. LacO repeats were integrated in the same position (i.e., <3 kbp from GAL1) for the DLDC and GFP cross-talk experiments. LacO repeats are immediately adjacent to the 5′ end of PES4 for the GAL/PES4 experiment. The DLDC and GAL/RPL9A cases were imaged in galactose and dextrose, whereas all other (control) cases were imaged in dextrose. Distances between loci and labels in schematic are not necessarily to scale.

Mentions: To conduct simultaneous tracking of the two GAL loci in diploid yeast, we labeled one copy with the LacO/LacI-GFP system and the other with the TetO/TetR-mCherry system (see Figure 2 for the labeling schematics described later). We collectively refer to this as a dual-label, dual-chromosome (DLDC) scheme. As one negative control, we also conducted experiments in which we simultaneously tracked only one copy of the GAL locus along with one copy of the gene PES4 located on chromosome VI, since both the nuclear positioning and transcriptional regulation of PES4 are unaffected by growth in galactose (Taddei et al., 2006). Although PES4 and the GAL locus can be considered transcriptionally independent, they are both located near the centromere of their respective chromosomes (∼50 and ∼40 kbp, respectively). Different pericentromeric genes have been shown to exhibit additional correlations during metaphase, related to their common association with the spindle pole body (Stephens et al., 2013). Although we attempted to exclude mitotic cells and include only cells in G1 interphase in our study, we still sought to exclude centromeric effects that could potentially arise due to the default Rabl-like conformation of budding yeast chromosomes. Therefore, as a second negative control, we examined correlations in a strain in which one copy of the GAL locus was labeled and one copy of the nonpericentromeric RPL9A gene on chromosome VII was labeled and studied this strain in both dextrose and galactose conditions.


Correlations of three-dimensional motion of chromosomal loci in yeast revealed by the double-helix point spread function microscope.

Backlund MP, Joyner R, Weis K, Moerner WE - Mol. Biol. Cell (2014)

Labeling scheme for each condition imaged. Green star corresponds to LacO/LacI-GFP label, and red star corresponds to TetO/TetR-mCherry label. TetO repeats were integrated at the same position relative to the GAL locus (<3 kbp from GAL1) wherever present. LacO repeats were integrated in the same position (i.e., <3 kbp from GAL1) for the DLDC and GFP cross-talk experiments. LacO repeats are immediately adjacent to the 5′ end of PES4 for the GAL/PES4 experiment. The DLDC and GAL/RPL9A cases were imaged in galactose and dextrose, whereas all other (control) cases were imaged in dextrose. Distances between loci and labels in schematic are not necessarily to scale.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4230621&req=5

Figure 2: Labeling scheme for each condition imaged. Green star corresponds to LacO/LacI-GFP label, and red star corresponds to TetO/TetR-mCherry label. TetO repeats were integrated at the same position relative to the GAL locus (<3 kbp from GAL1) wherever present. LacO repeats were integrated in the same position (i.e., <3 kbp from GAL1) for the DLDC and GFP cross-talk experiments. LacO repeats are immediately adjacent to the 5′ end of PES4 for the GAL/PES4 experiment. The DLDC and GAL/RPL9A cases were imaged in galactose and dextrose, whereas all other (control) cases were imaged in dextrose. Distances between loci and labels in schematic are not necessarily to scale.
Mentions: To conduct simultaneous tracking of the two GAL loci in diploid yeast, we labeled one copy with the LacO/LacI-GFP system and the other with the TetO/TetR-mCherry system (see Figure 2 for the labeling schematics described later). We collectively refer to this as a dual-label, dual-chromosome (DLDC) scheme. As one negative control, we also conducted experiments in which we simultaneously tracked only one copy of the GAL locus along with one copy of the gene PES4 located on chromosome VI, since both the nuclear positioning and transcriptional regulation of PES4 are unaffected by growth in galactose (Taddei et al., 2006). Although PES4 and the GAL locus can be considered transcriptionally independent, they are both located near the centromere of their respective chromosomes (∼50 and ∼40 kbp, respectively). Different pericentromeric genes have been shown to exhibit additional correlations during metaphase, related to their common association with the spindle pole body (Stephens et al., 2013). Although we attempted to exclude mitotic cells and include only cells in G1 interphase in our study, we still sought to exclude centromeric effects that could potentially arise due to the default Rabl-like conformation of budding yeast chromosomes. Therefore, as a second negative control, we examined correlations in a strain in which one copy of the GAL locus was labeled and one copy of the nonpericentromeric RPL9A gene on chromosome VII was labeled and studied this strain in both dextrose and galactose conditions.

Bottom Line: As controls, we tracked pairs of loci along the same chromosome at various separations, as well as transcriptionally orthogonal genes on different chromosomes.This relative increase has potentially important biological implications, as it might suggest coupling via shared silencing factors or association with decoupled machinery upon activation.We also found that on the time scale studied (∼0.1-30 s), the loci moved with significantly higher subdiffusive mean square displacement exponents than previously reported, which has implications for the application of polymer theory to chromatin motion in eukaryotes.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Stanford University, Stanford, CA 94305.

Show MeSH
Related in: MedlinePlus