Display Settings:

Items per page

Results: 10

1.
FIG. 1.

FIG. 1. From: Drosophila Nipped-B Protein Supports Sister Chromatid Cohesion and Opposes the Stromalin/Scc3 Cohesion Factor To Facilitate Long-Range Activation of the cut Gene.

Nipped-B protein is widely expressed and is in the nucleus. Cultured S2 cells, blastoderm embryos, third-instar salivary glands, and third-instar fat bodies were immunostained for Nipped-B, DNA (DAPI), and in some cases nuclear lamin. The third-instar salivary gland micrographs were obtained with a confocal microscope. The cultured cells in the second row from the top were treated with Nipped-B RNAi for 48 h prior to being stained. The bars in the left-hand column are 5 μm. The photographs of the Nipped-B immunostaining of S2 cells treated with Nipped-B RNAi and the blastoderm metaphase embryo were not adjusted for contrast because it could not be unambiguously determined if the low overall staining is authentic or background. The contrast was adjusted in the other photographs to show the detail of the strong nuclear Nipped-B staining.

Robert A. Rollins, et al. Mol Cell Biol. 2004 April;24(8):3100-3111.
2.
FIG. 7.

FIG. 7. From: Drosophila Nipped-B Protein Supports Sister Chromatid Cohesion and Opposes the Stromalin/Scc3 Cohesion Factor To Facilitate Long-Range Activation of the cut Gene.

Rad21/Scc1 RNAi and a heterozygous Nipped-B407 mutation combine to reduce viability. Females of the Nipped-B and P{Sym-pUAST-Rad21/Scc1}-2 genotypes were produced by crossing y w ctK; P{Act5c-Gal4}/CyO y+ females to appropriate males at 25°C. The genotypes of the males were as follows: left, y w; P{Sym-pUAST-Rad21/Scc1}-2; middle, y w; Nipped-B407 P{mini-w+}57B/CyO Kr y+; right, y w; Nipped-B407 P{mini-w+}57B/CyO Kr y+; P{Sym-pUAST-Rad21/Scc1}-2. Viability was calculated by dividing the number of Gal4+ (y; Cy+) adult female progeny of the indicated genotype by the number of Gal4 (y+; Cy) progeny of the same genotype. The presence of the Nipped-B407 chromosome in the progeny was confirmed by the 57B P insertion, which displays variegated mini-white gene expression (48). The error values are 95% confidence intervals (±2 standard errors [s.e.]). The green bars represent crosses in which Rad21/Scc1 RNAi was induced.

Robert A. Rollins, et al. Mol Cell Biol. 2004 April;24(8):3100-3111.
3.
FIG. 6.

FIG. 6. From: Drosophila Nipped-B Protein Supports Sister Chromatid Cohesion and Opposes the Stromalin/Scc3 Cohesion Factor To Facilitate Long-Range Activation of the cut Gene.

Nipped-B and Rad21/Scc1 RNAi increase SA/Scc3 mRNA levels. Control males (y w) or males with the P{Sym-pUAST} RNAi inserts were crossed to females with the hsp70-Gal4 driver at 25°C, as diagrammed at the top. Total RNA was isolated from Gal4+ third-instar progeny. Northern blots (5 μg per lane) were sequentially probed for SA/Scc3 mRNA (3.8 kb) and rp49 (0.9 kb). The graph shows the PhosphorImager quantification of SA/Scc3 mRNA normalized to the rp49 loading control with the value for the y w control (yellow bar) set to 100. The average value for the three Nipped-B RNAi inserts (red bars) is 200 ± 10 (95% confidence interval), and the average value for the three Rad21/Scc2 RNAi inserts (green bars) is 140 ± 20.

Robert A. Rollins, et al. Mol Cell Biol. 2004 April;24(8):3100-3111.
4.
FIG. 5.

FIG. 5. From: Drosophila Nipped-B Protein Supports Sister Chromatid Cohesion and Opposes the Stromalin/Scc3 Cohesion Factor To Facilitate Long-Range Activation of the cut Gene.

SA/Scc3 RNAi reduces SA/Scc3 mRNA and increases Rad21/Scc1 mRNA. Crosses to females with the Act5c-Gal4 driver were conducted at 25°C, as diagrammed at the top, with y w control males and males with three independent inserts of the P{Sym-pUAST-SA/Scc3} transposon, as indicated at the bottom. Total RNA was isolated from the Gal4+ and Gal4 third-instar progeny. Northern blots (5 μg of RNA per lane) were sequentially probed for SA/Scc3 (3.8-kb), Rad21/Scc1 (2.3-kb), and rp49 (0.9-kb) transcripts. (Top) Level (in arbitrary PhosphorImager units) of SA/Scc3 mRNA normalized to rp49 loading control. (Middle) Level of Rad21/Scc1 2.3 mRNA normalized to rp49. (Bottom) Ratio of SA/Scc3 transcript to Rad21/Scc1 for each lane, after the ratio for the y w control was set to 1.0. The blue bars indicate the presence of SA/Scc3 RNAi, and the yellow bars represent the control cross. The solid bars represent Gal4 siblings from the RNAi crosses.

Robert A. Rollins, et al. Mol Cell Biol. 2004 April;24(8):3100-3111.
5.
FIG. 4.

FIG. 4. From: Drosophila Nipped-B Protein Supports Sister Chromatid Cohesion and Opposes the Stromalin/Scc3 Cohesion Factor To Facilitate Long-Range Activation of the cut Gene.

Nipped-B RNAi reduces Nipped-B mRNA levels. Crosses were conducted with three independent insertions of the P{Sym-pUAST-Nipped-B} RNAi transposon at 25°C and with an Act5c-Gal4 driver, as shown at the top. (Top) Northern blot of total RNA (5 μg per lane) prepared from third-instar larvae probed with an antisense [32P]RNA probe that detects both the 7.0-kb Nipped-B mRNA and the 2.2-kb Sym-pUAST-Nipped-B sense strand. The 4-kb transcript (asterisk) is a minor product of Sym-pUAST-Nipped-B. RNA was prepared from Gal4+ and Gal4 siblings for each RNAi insertion (numbered 1, 2, and 3), as indicated at the bottom. (Middle) PhosphorImager quantification (in arbitrary units) of the Nipped-B mRNA normalized to PhosphorImager values obtained for rp49 transcripts (0.9 kb) as a loading control after reprobing of the same Northern blot. (Bottom) Normalized PhosphorImager quantification of the Sym-pUAST-Nipped-B sense transcript. The red bars represent progeny in which Nipped-B RNAi was induced (Gal4+), and the solid bars indicate siblings in which Nipped-B RNAi was not induced (Gal4).

Robert A. Rollins, et al. Mol Cell Biol. 2004 April;24(8):3100-3111.
6.
FIG. 2.

FIG. 2. From: Drosophila Nipped-B Protein Supports Sister Chromatid Cohesion and Opposes the Stromalin/Scc3 Cohesion Factor To Facilitate Long-Range Activation of the cut Gene.

Nipped-B mutant second-instar larval neuroblasts display high levels of PSCS. The photographs are examples of wild-type and Nipped-B mutant second-instar brain metaphases. The Nipped-B mutant metaphases range from those that have no visible defects (left) to those in which all chromosomes display PSCS (right). The table below summarizes data obtained with several genotypes. Large numbers of metaphases with second-instar larvae were difficult to obtain, particularly with Nipped-B mutants, but the PSCS frequency (freq) in the second-instar wild-type control is close to that observed with third-instar neuroblasts treated with colchicine and hypotonic solution (33). The second column from the left is the number of metaphases scored, and the third column is the number of scored metaphases that had at least one chromosome with PSCS. For the larger samples, the frequency and the 95% confidence intervals were calculated based on the frequency and sample size. The fourth and fifth columns are the number of metaphases that were hypoploid or hyperploid, respectively. Only a single chromosome was affected in the wild-type metaphases that displayed PSCS, while more than one chromosome was affected in most of the Nipped-B mutant metaphases that displayed PSCS.

Robert A. Rollins, et al. Mol Cell Biol. 2004 April;24(8):3100-3111.
7.
FIG. 3.

FIG. 3. From: Drosophila Nipped-B Protein Supports Sister Chromatid Cohesion and Opposes the Stromalin/Scc3 Cohesion Factor To Facilitate Long-Range Activation of the cut Gene.

Nipped-B and Cohesin RNAi reduce adult viability. Crosses with Act5c-Gal4 females were conducted at 25°C, as diagrammed above the graph, with y w control males and males with the P{Sym-pUAST} RNAi inserts indicated at the bottom. Because hemizygous ctK males display reduced viability (∼25% that of the wild type), only adult females were used to calculate the effect of RNAi on viability. More than 100 progeny females were scored for each cross. The RNAi inserts are transcribed in Gal4+ flies. To calculate the effect of RNAi on viability, the ratio of Gal4+ (y; Cy+) progeny was divided by the number of Gal4 (y+; Cy) progeny ([Gal4+/Gal4−]). This value was then divided by the Gal4+/Gal4 ratio obtained in the control cross ([Gal4+/Gal4−]yw) to account for the small negative effect of the CyO balancer chromosome on the viability of Gal4 flies. The y w control viability (yellow bar) is thus set to 100%. All three Nipped-B RNAi inserts strongly reduced viability (0 to 7%; red bars). The three Rad21/Scc1 RNAi inserts displayed more variable effects (25 to 108% viability; green bars), and the three SA/Scc3 RNAi inserts were more lethal (0 to 35% viability; blue bars).

Robert A. Rollins, et al. Mol Cell Biol. 2004 April;24(8):3100-3111.
8.
FIG. 9.

FIG. 9. From: Drosophila Nipped-B Protein Supports Sister Chromatid Cohesion and Opposes the Stromalin/Scc3 Cohesion Factor To Facilitate Long-Range Activation of the cut Gene.

SA/Scc3 RNAi decreases the severity of the ctK wing-nicking phenotype. Crosses were conducted at 27°C with an hsp70-Gal4 driver with three independent insertions of the Cohesin subunit RNAi transposons. Both a y w control lacking an RNAi transposon (yellow bar) and a control with an RNAi transposon directed against the CG4203 non-Cohesin gene (dark-blue bar) were performed. At least 30 ctK Gal4+ male progeny were counted for each cross, and the distributions are presented in standard box plots as described for Fig. 8. The asterisks indicate distributions significantly different from both controls in pairwise t tests. None of the RNAi insertions significantly affected viability (93 to 103% of the Gal4+/Gal4 adult female ratio observed in the y w control). The median wing nick value for the y w control in this experiment was lower than in Fig. 8 because the higher temperature partially suppressed the ctK wing margin phenotype. Green bars, Rad21/Scc1 RNAi; light blue bars, SA/Scc3 RNAi.

Robert A. Rollins, et al. Mol Cell Biol. 2004 April;24(8):3100-3111.
9.
FIG. 10.

FIG. 10. From: Drosophila Nipped-B Protein Supports Sister Chromatid Cohesion and Opposes the Stromalin/Scc3 Cohesion Factor To Facilitate Long-Range Activation of the cut Gene.

Proposed model to explain opposing effects of Nipped-B and SA/Scc3 Cohesin subunit on cut gene expression. The central proposals are that Nipped-B both loads and removes Cohesin from the region between the wing margin enhancer and the promoter and that Cohesin acts as a boundary or insulator to block enhancer-promoter communication. In a simple version of this model, Nipped-B acts as an equilibrium factor by opening the Cohesin ring and allowing either loading or removal of Cohesin from the chromosome. Partial reduction of Nipped-B, either in heterozygous Nipped-B mutants or by RNAi, would not alter the equilibrium endpoint, and thus would not have a significant effect on sister chromatid cohesion, but would decrease the frequency at which Cohesin could be removed to allow long-range activation. In homozygous Nipped-B mutants, in which Nipped-B activity is strongly reduced, binding of Cohesin to chromosomes would be reduced, resulting in sister chromatid cohesion defects as shown in Fig. 2. This model is consistent with the yeast homologues of Nipped-B supporting sister chromatid cohesion by loading Cohesin and with the requirement for Cohesin components at the yeast HMR chromatin domain boundary discussed in the text. The mechanisms by which the yeast Nipped-B homologues facilitate chromosomal binding of Cohesin are unknown; direct contact between Nipped-B and Cohesin to open the Cohesin ring is depicted for simplicity.

Robert A. Rollins, et al. Mol Cell Biol. 2004 April;24(8):3100-3111.
10.
FIG. 8.

FIG. 8. From: Drosophila Nipped-B Protein Supports Sister Chromatid Cohesion and Opposes the Stromalin/Scc3 Cohesion Factor To Facilitate Long-Range Activation of the cut Gene.

Nipped-B RNAi increases the severity of the ctK wing-nicking phenotype. Crosses with a y w control and two independent insertions of the P{Sym-pUAST-Nipped-B} transposon were conducted at 25°C with an hsp70-Gal4 driver, as diagrammed at the top. Wing margin nicks in Gal4+ adult males were counted, and the distributions of wing nicks per fly for each cross are summarized in standard box plots. At least 30 ctK Gal4+ male progeny were scored for each cross. For each cross, the bottom edge of the colored box represents the 25th percentile (i.e., 25% of the values were lower) and the top edge represents the 75th percentile. The horizontal line across the colored portion of each box indicates the median value (50th percentile) for that cross. The crossbars at the ends of the lines extending from each box represent the 10th (bottom) and 90th (top) percentiles. A dashed line across the entire graph representing the median value for the y w control makes it easier to compare the distributions. For example, in the cross with the P{Sym-pUAST-Nipped-B}-1 insert, 75% of the male progeny had wing nick values above the median control value, and with P{Sym-pUAST-Nipped-B}-2, 90% had more wing nicks than the control median. The asterisks indicate distributions that differ significantly from the control y w cross distribution in pairwise t tests. Adult Gal4+ female viability rates in the Nipped-B RNAi crosses were 68 and 72% of the control viability rates for insertions 1 and 2, respectively. Yellow bar, y w control; red bars, Nipped-B RNAi.

Robert A. Rollins, et al. Mol Cell Biol. 2004 April;24(8):3100-3111.

Display Settings:

Items per page

Supplemental Content

Recent activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...
Write to the Help Desk