Lab No ( 3 )

Polytene Chromosomes

Polytene chromosomes are giant chromosomes
common to many dipteran (two-winged) flies.

They begin as normal chromosomes, but through
repeated rounds of DNA replication without any cell
division (called endoreplication), they become large,
banded chromosomes.

For unknown reasons, the centromeric regions of the
chromosomes do not endoreplicate very well. As a
result, the centromeres of all the chromosomes bundle
together in a mass called the chromocenter.






The salivary gland cells in the larval stages of
Drosophila contain large, multistranded polytene
chromosomes.

This laboratory procedure outlines the
mechanism by which salivary glands are
removed and prepared so that the polytene
chromosomes may be observed.

Drosophila melanogaster is easier to dissect and
remove the salivary glands from the larvae of
this species.
Polytene
Chromosomes
from Salivary
Glands
Student Handout
Materials
For each student group:
compound microscope
2 teasing needles
insect pins
microscope slides
cover glasses
stock supply of Drosophila melanogaster larvae
dropping bottle of aceto-orcein stain
dropping bottle of 45% acetic acid solution
Procedures

Removing the salivary glands:
1- Remove a large larva from the stock of D.
melanogaster. Larger larvae are easier to
dissect. However, select an active larva and one
that has not started to pupate.
2- Using the compound microscope, dissect the
larva by placing one teasing needle on the
posterior aspect of the larva and the other
needle at the anterior end, near the black mouth
parts.
3- Carefully pull outward with the anterior needle.

Procedure for removing Drosophila salivary glands
( cont )
4- There are two transparent salivary glands
located anteriorly in the larva.

5- Discard all of the larva except for the
salivary glands.
Staining and Observing

1- Place 2 drops of aceto-orcein stain on the
salivary glands, and let it stand for 10 minutes.
2- Place a cover slip over the glands, and using
your thumb and a paper towel, push down on
the slide. The pressure applied will squash the
glands, rupture the nuclear membrane, and free
the chromosomes.
3- Using a compound microscope, observe the
slide under low and high magnification.
4- Make the slide permanent by brushing along the
edges of the cover slip with clear nail polish

The Puffing
Puffing is the term that describes structural
changes in polytene chromosomes. If one
observes polytene chromosomes during
the late prepupal stage, different bands
appear to be puffed up. For 40 years, this
has been understood to be the result of
gene activity .

Definition of polytene chromosome
A giant chromosome produced by an endomitotic process
in which, following synapsis of the two homologues,
multiple rounds of replication produce chromatids that
remain synapsed together in a haploid number of
chromosomes. Polytene chromosomes have been studied
mostly in Drosophila salivary glands, in which
chromosomes undergo 10 cycles of replication without
separation of the daughter chromosomes. This leads to
1024 identical strands of chromatin aligned side by side.
These chromosomes are easy to see with a light
microscope because of their large size and precise
alignment. The chromosomes are seen as distinct
alternating dark and light bands which has given rise to a
polytene chromosome map. The dark bands correspond to
more condensed regions of the chromatin, and the light
(interband) regions are less dense regions.