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