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PLNT3140 INTRODUCTORY CYTOGENETICS    

Assignment 1

Sept. 28, 2017

This assignment is worth 5% of the course grade.

Due: Tue. Oct. 10, 2017.


Chromosome pairing normally occurs only during meiosis. However, in a number of species the phenomenon of somatic homologue pairing has been observed, in which chromosomes pair even in somatic cells. Flourescence In-situ Hybridization was done to examine somatic pairing in Drosophila melanogaster. The authors reasoned that in diploid cells, if any part of a chromosome was paired with its homologue, only one spot should be seen rather than the two spots normally seen in diploid cells where homologue pairing does not occur. In cell line Kc167, most cells are tetraploid (4N) rather than diploid, so you would expect to see between 1 and 4 signals per nucleus, depending on how many of the chromosomes were paired at this locus. This approach for observing whether part of a chromosome is paired or unpaired was used in the following experiments.

FISH probes were prepared from six loci on chromosomes X, II and III (A) . Probes were hybridized to chromosomes in Drosophila tissue culture cells. An example using probe 28B1 is shown  in (B). Two images were made, one using DAPI dye that stains all DNA, and the other using the 28B1 probe. The images were merged to show the positions of signal in each nucleus.



1. (2 points) Experiments were performed using probes for both euchromatic regions and heterochromatic regions of chromosomes. Summarize the main conclusions that you can draw from these observations.

Euchromatic loci

E - Percentage of nuclei showing 1, 2, 3 or 4 signals per nucleus.

F - For each locus (probe), distances between spots for nuclei visualized E,  as a percentile plot.

 
 

Distance - All inter-signal distances within each nucleus are ranked, shortest to longest, left to right, where 100% corresponds to the greatest distance observed. Signals separated by <= 0.5 Ám (----) are considered one signal.
Percentile plot - Cumulative percentage of all observations, ranked from smallest to largest. Example: for locus 69C2 80 percent of the observed distances are 1 Ám or less; 90 % of observed distances are 2
Ám or less; 95% of observed distances are 4 Ám or less.
Heterochromatic loci

G- Percentage of nuclei showing 1, 2, 3 or 4 signals per nucleus.

H - The nuclei as in G, as a percentile plot for each probe.


2. (2 points) A computer simulation was done in which four signals (to represent the four copies of a chromosome in tetraploid cells) were placed at random positions in nuclei.

A - Distances (Ám) between signals for 28B1 loci within a nucleus. A percentile plot for randomly-placed signals is shown in the black curve. Experimentally observed distances between 28B1 loci are also plotted for comparison.

B - Experimentally observed distances between 28B1 loci and non-homologous loci 21E3, or 69C2, compared to computer simulation.




What do you think the authors were trying to show, from this simulation?


3. (1 point) Tissue culture cells were separated into different fractions using a Fluorescence Activated Cell Sorter. Because cells in G1, S and G2 phases have different amounts of DNA (see B, inset), cells labelled with the DNA-specific dye Hoechst 33342 can be separated into 3 samples, each containing a population of cells from these three phases of the cell cycle. (Since mitotic cells tend to be a small percentage of the population, we can ignore the fact that the G2 sample would also be expected to include a small amount of M-phase cells.)

Nuclei were scored for the number of signals per nucleus for probe 28B1.

What was the purpose of this experiment, and what can you conclude from it?

Submitting your assignment

Note on grading: In assigning a grade, some consideration may be given to how the answer communicates your ideas. Keep in mind the following:

Note on academic integrity: The results in this assignment obviously are derived from the research literature. It will be considered a breach of academic integrity to search for the paper on the Internet and simply copy the author's conclusions from the paper.

 Notes: If you have questions, it may help to send me a messge at frist@cc.umanitoba.ca.