Procedure of observation of the polytene chromosome in drosophila in the lab.

1. STUDY OF POLYTENE
CHROMOSOME
POLYTENE CHROMOSOMES IN DROSOPHILA

2. INTRODUCTION
• Polytene chromosomes are over-sized chromosomes which have
developed from standard chromosomes and are commonly found in the
salivary glands of DROSOPHILA MELANOGASTER Specialized cells
undergo repeated rounds of DNA REPLICATION without CELL DIVISION,
to increase CELL volume, forming a giant polytene chromosome.
Polytene chromosomes form when multiple rounds of replication
produce many sister chromatids that remain synapsed together.

3. HISTORY
• Polytene chromosomes were originally observed in the larval SALIVARY
GLANDS of CHIROMOMUS midges by Balbiani in 1881, but the
hereditary nature of these structures was not confirmed until they were
studied in DROSOPHILA MELANGOSTER in the early 1930s by EMIL
HEITZ and HANS BAUER. They are known to occur in secretory tissues of
other DIPTERAN insects such as the MALPHIGIAN TUBULES of Sciara and
also in protists, plants, mammals, or in cells from other INSECTS. Some
of the largest polytene chromosomes described thus far occur in larval
salivary gland cells of the Chironomid genus Axarus.

4. FUNCTIONS
• In addition to increasing the volume of the cells' nuclei and causing cell
expansion, polytene cells may also have a metabolic advantage as
multiple copies of genes permits a high level of gene expression.
In Drosophila melanogaster, for example, the chromosomes of the larval
salivary glands undergo many rounds of endoreduplication, to produce
large amounts of glue before pupation. Another example, within the
organism itself is the tandem duplication of various polytene bands
located near the centromere of the X chromosome which results in the
Bar phenotype of kidney-shaped eyes.

5. STRUCTURE
• Polytene chromosomes have characteristic light and dark banding
patterns that can be used to identify chromosomal rearrangements and
deletions. Dark banding frequently corresponds to inactive chromatin,
whereas light banding is usually found at areas with higher
transcriptional activity. The banding patterns of the polytene
chromosomes of Drosophila melanogaster were sketched in 1935
by Calvin B. Bridges.

8. STRUCTURE CONTINUED..
• The banding patterns of the chromosomes are especially helpful in
research, as they provide an excellent visualization of transcriptionally
active chromatin and general chromatin structure. For example, the
polytene chromosomes in Drosophila have been used to support the
theory of genomic equivalence, which states that all of the cells in the
body maintain the same genome. Polytene chromosomes are about
200 micrometer in length. The chromonema of these chromosomes
divide but do not separate. Therefore, they remain together to become
large in size.

9. PROCEDURE
• The success of these experiments is largely determined by the
developmental stage of the larvae.
• If they are too small, their salivary glands will not be sufficiently
developed to yield good chromosomes.
• Choose the largest larvae that you can find that is still moving actively.
• Do not choose the larvae that is noticebly darker than the rest.
• Darkening of the cuticle is a sign that pupation is imminent.

10. PREPARING SALIVARY SQUASHES
• Place a drop of 0.7% saline on a clean slide.
• Transfer an appropriate larva to the slide and place it on the stage of a dissecting
microscope.
• Using the enhanced detail afforded by the microscope, firmly grasp the posterior end
of the larva with forceps and use a dissecting needle to pierce through the head.
• Using a continuous motion pull the needle away from the body.
• The salivary glands are recognised by the foll. features:
• They are paired and each is identical in shape and size.
• They have a glistening, translucent appearance.
• Each gland should have an opaque fat body associated with it.

11. • While continuing to observe under the microscope, separate the salivary
glands from any extraneous material, such as the fat bodies or parts of
the digestive tract.
• Once cleaned, remove the used saline and debris from the slide. Add
fresh saline and allow the glands to soak for ten minutes.
• Carefully place 1-2 drops of acetocarmine or aceto-orcein stain on each
of the regions.
• Wait for a couple of minutes and proceed to the next step.

12. • Crushing glands to expose cells: Place a cover slip carefully in each of
the regions of the slide where salivary glands were placed.
• Crush each cover slip against the slide with the help of your thumb.
• If the crushed region appears too thick, flatten it further by tapping the
coverslip multiple times with the back of a dissecting needle
• Allow the exposed cells to absorb the stain for 5-10 minutes.

13. IDENTIFYING THE POLYTENE
CHROMOSOME UNDER A MICROSCOPE
• Place the slide under the microscope .
• Locate the squashed regions under 4x and 10x objective magnification
to see where to focus.
• Once those are determined change the objective to 40x to locate
individual cells.
• If certain fibrous projections appear, switch to 100x.
• Light and dark bands are observed as shown.
• Hence the procedure comes to an end.

14. REFERENCES
• https://en.wikipedia.org/wiki/Polytene_chromosome
• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2821744/
• http://msg.ucsf.edu/sedat/polytene_chrom.html