POLYTENE CHROMOSOMES & CHROMOSOME
To stain and identify the polytene chromosomes of the salivary glands
of Drosophila melanogaster. To induce puffing, which is a
visual indicator of RNA transcription.
Many larval and some adult tissues of
insects in the family Diptera are characterized by nuclei with giant
chromosomes. These chromosomes develop by multiple replications of the
chromosomes within each cell during development. Each nucleus will
contain hundreds of copies of each chromosome. Cells are considered
polyploid if they have more than two copies of each chromosome. If the
chromosomes align perfectly forming large cables of chromosomes they
In Drosophila melanogaster,
chromosomes of the larval salivary gland contain about 1024 copies of
the DNA, or ten doublings from the normal 2n condition, of each of the
three chromosomes. Each gene is exactly aligned with its homologs on
the other 1023 copies. The pattern of condensed regions
(heterochromatin), and transcribed regions (euchromatin) gives a
series of about 5000 light and dark bands when the chromosomes are
stained with orcein. The banding patterns of the chromosomes show
significant phylogenetic and ontogenetic stability. Genetic maps
relate these bands to their functions. In general, the DNA in each
band codes for a single function, although there are exceptions to
this observation. Drosophila has given us substantial insight into DNA
function and gene organization. In March 2000, the full genome map of
D. melanogaster was published in the journal Science.
At certain times during development,
some bands undergo a reversible modification to form structures known
as "puffs." Puffs are localized expansions of the polytene chromosome
structures. Puffs are sites of synthesis of RNA and result from the
activation of the gene (or genes) contained within a particular band.
This means that changes in gene activity can be observed
microscopically as changes in polytene chromosome puffing pattern. The
size of the puff reflects the number of copies of that gene that are
being transcribed - larger puffs have a larger percentage of the 1024
chromatids being simultaneously copied into mRNA. The specific RNAs
are translated into a set of polypeptides
Puffing patterns that occur during
normal development can sometimes be induced by experimental
treatments. Changes in the patterns of puffing occur as a direct
consequence of changes in the concentration of the insect's growth and
molting hormone, ecdysone. Initial changes in puffing can be seen in
salivary glands exposed to ecdysone for less than 15 minutes. The
pattern of gene activation starts with new transcription of a few
"early" genes whose products in turn regulate a set of "late" genes.
This regulation can include increased or decreased synthesis as well
as initiation or termination of transcription.
Increased or decreased transcription
can also result from a wide variety of environmental factors including
a brief heat shock. In 1962, heat shock was found to induce a unique
set of puffs and the tranlation of the heat shock polypeptides or "hsp's".
When Drosophila larvae, or their excised tissues, are subjected to a
brief heat shock (for example, 40 min. at 37o C, the normal culture
temperature being 25o C), puffs are induced at a few specific sites.
In D. melanogaster there are nine heat inducible puffs, in a
related species, D. hydei, there are six. The induction of the
puffs by the heat shock is very rapid; it occurs within one minute of
the temperature increase though the puffs continue to increase in size
for some 30 to 40 min (at 37o C) before regressing. The maximum sizes
of the induced puffs are a function of the severity of the temperature
shock, at least until lethal temperatures are met. The induction
requires RNA, but not protein synthesis. However, in the absence of
protein synthesis the induced puffs fail to regress unless the
temperature is returned to normal. Prolonged temperature shock (e.g.,
more than 1 hour) results in additional changes in puffing activity;
all other puffs active at the time the temperature shock commenced
regress. The heat shock response of specific RNA and protein synthesis
occurs in all tissues of the fly and has been observed in permanent
tissue culture cell lines.
In this experiment, you will dissect
out the salivary glands of
Dissecting pins (I prefer insect pins over dissecting probes.)
Glass slides and cover slips
45% acetic acid
Use mature third instar larvae - the
fattest crawling larvae in the small petri dishes. If the larvae have
started to form the puparium, the fat body quickly disappears to make
room for the adult salivary gland.
1. Adjust your dissecting microscope
so it has a dark background or dark field where the light is coming
from an angle so you can see the tissues clearly.
2. Dissect out the salivary glands
in 45% acetic acid. Glands should remain in acetic acid for relatively
short periods of time. Do not allow the glands to dry.
Place pins near head (region where dark mouth parts are located) and
about half way down the body, then pull pins apart.
- If you pull
gently, the glands will be easy to find between the anterior and
posterior sections. If the gut and other tissues just form a lump,
it is almost impossible to find the glands. Start again.
- Once you see the
salivary glands, dissect away the abdomen.
- Be sure
to remove the head. Don't try to remove
the fat bodies that are located along the salivary glands. You will
see the fatty material under the slide, but it will not interfere
with viewing the glands or staining their chromosomes. The mouth
parts are very hard and if they are under the coverslip, you will
not be able to put enough pressure on it to crush them. They are
much thicker than the cells of the salivary gland, the cells and
their nuclei will not be ruptured.
3. Carefully remove most of the drop
of acetic acid by tilting the slide and removing the drop with a
kimwipe. Keep the kimwipe away from the gland itself. Add one drop of
1 N HCl for 30 seconds then carefully remove.
4. Quickly rinse the gland with
lacto-acetic acid to prevent precipitation of stain.
5. Add one drop of stain and cover
slide with half of a petri dish. Wait 15 minutes.
6. Rinse stained glands with
lacto-acetic acid. Place cover slip over gland and put it between
layers of paper toweling. Press firmly on the cover slip, taking care
that it does not slip. You may need to prepare two or three slides
before you master the skill of spreading the chromosome arms without
tearing the chromosomes.
7. Dissect a second gland and repeat
staining in case the first is not successful.
8. Examine the chromosomes. Adjust
your microscope for Kohler illumination and look for large pinkish
nuclear regions. If you look carefully along the length of each
chromosome, you may see puffs, genes that are being transcribed.
9. If you have a copy of the
Drosophila chromosome map handy, you can attempt to identify
chromosomes 1, 2, 3, and 4.
Lacto-acetic acid: 85% lactic acid: 60%
acetic acid: deionized water 1:1:1
Lacto-aceto-orcein stain: 1% orcein (synthetic) in equal volumes of
85% lactic acid and 60% acetic acid.
Heat the mixture to boiling and boil a few minutes.
Cool and filter. Stain may need to be filtered if kept for prolonged
Acetic acid should be prepared fresh every few days.
Modified from a laboratory procedure
by Jeanette Holden, formerly of Queen's University, Kingston, Ontario.