Net64tax: Створена сторінка: Stretched bivalents were defined as the chromosomes that reach >70% of their maximal interkinetochore distance. If a stretched bivalent maintained the angle wit...

2017-03-21T21:05:44Z

Створена сторінка: Stretched bivalents were defined as the chromosomes that reach >70% of their maximal interkinetochore distance. If a stretched bivalent maintained the angle wit...

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Stretched bivalents were defined as the chromosomes that reach >70% of their maximal interkinetochore distance. If a stretched bivalent maintained the angle with the estimated spindle axis within [http://www.selleckchem.com/products/cpi-1205.html Epigenetics inhibitor] when the diameter of the disassembling nucleolus becomes less than 5?��m. The other is time after metaphase entry, which is defined as the time when half of chromosomes established orientation along the spindle axis. All calculations were automated by an in-house developed Java (Sun Microsystems) program and R (http://www.r-project.org/). The 2D and the 3D plots were generated by the plotting software Gnuplot (http://www.gnuplot.info/) or Prism (GraphPad) and the ray-tracing software Pov-Ray (http://www.povray.org/), respectively, [http://www.selleckchem.com/products/mk-4827-niraparib-tosylate.html Selleckchem MK-4827] controlled by scripts generated from the Java program. Extended Experimental Procedures Culture of Oocytes and Microinjection Oocytes were collected from ?8-week-old FVB mice according to the guidelines of EMBL Laboratory Animal Resources and cultured as described (Schuh and Ellenberg, 2007). Microinjection of in?vitro-transcribed RNAs to the oocytes was performed (Jaffe and Terasaki, 2004) with 1pl EGFP-CENP-C, 0.2pl mCherry-H2B, 1pl 3mCherry-CENP-C, 2pl EGFP-MAP4, 1pl Mad2-EGFP of 1?��g/��l mRNAs. Time-lapse image acquisitions were performed using a customized Zeiss LSM510 Meta confocal microscope equipped with the 40x C-Apochromat 1.2NA water immersion objective lens (Carl Zeiss) with an in-house-developed 3D multilocation tracking macro (Rabut and Ellenberg, 2004). We recorded the subvolume centered around chromosomes or kinetochores by 3D time-lapse (4D) imaging for 3�C5 cells in parallel. For kinetochore tracking with EGFP-CENP-C, we imaged 17 z-confocal sections (every 1.5 or 2.0?��m) of 256?�� 256 or 512?�� 512 pixel xy images, in total covering at least 32.0?��m �� 32.0?��m �� 25.5?��m volume, at time interval of 90?s for 9?hr after induction of maturation, covering NEBD to anaphase onset of meiosis I. For 3mCherry-CENP-C, we imaged 17 z-confocal sections (every 2.0?��m) of 256?�� 256 pixel xy images, in total covering a 37.5?��m �� 37.5?��m �� 34.0?��m volume, at time interval of 90?s from 1?hr to 6?hr after NEBD, covering [http://en.wikipedia.org/wiki/DEF6 DEF6] phase 2, phase 3, and the first part of phase 4. For peak enhancement and background subtraction for kinetochore signals, we subtracted the kinetochore images processed by a ten-pixel Gaussian blur from the same images processed by a two-pixel Gaussian blur. We reversed the time sequence of the movie because it was more error-free to perform kinetochore tracking reversely from late metaphase, when homologous kinetochore pairs are obvious because of chromosome biorientation. These image processing tasks were automated by an in-house developed macro in ImageJ.

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Net64tax: Створена сторінка: Stretched bivalents were defined as the chromosomes that reach >70% of their maximal interkinetochore distance. If a stretched bivalent maintained the angle wit...