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Sem1_P4

The document outlines a procedure for preparing polytene chromosomes from the salivary glands of Drosophila larvae, including the isolation of glands and staining techniques. Polytene chromosomes are characterized by their large size and distinct banding patterns, which are useful for analyzing gene expression and confirming genetic maps. The procedure involves specific reagents and careful handling of the larvae to ensure successful chromosome preparation.

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5 views5 pages

Sem1_P4

The document outlines a procedure for preparing polytene chromosomes from the salivary glands of Drosophila larvae, including the isolation of glands and staining techniques. Polytene chromosomes are characterized by their large size and distinct banding patterns, which are useful for analyzing gene expression and confirming genetic maps. The procedure involves specific reagents and careful handling of the larvae to ensure successful chromosome preparation.

Uploaded by

yaragollanaagaanandini
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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P4.

Preparation of Polytene chromosome from Drosophila salivary gland

Aim : To Prepare squashes of polytene chromosomes of the salivary gland of a wild-type strain,
stain them, and analyze the chromosomes and their banding pattern.

Objectives
1. Isolation of salivary glands
2. Preparation of Polytene chromosomes

Theory:

 Balbiani first discovered a structure in the nuclei of secretory glands of midges


 Painter, Heitz and Bauer, rediscovered them in the salivary gland of Drosophila and
recognised them as a chromosome
 These are called polytene by Kollar due to the presence of many chromonemata in them
 Chromosome puffs or Balbiani rings are present, which are the swelling of bands due to
DNA unfolding into open loops. These are the region of the intense transcription or mRNA
formation
Cells from the larval salivary gland of Drosophila contain chromosomes that are about 100 times
thicker than the chromosomes found in most other cells of the organism. During larval
development, these cells cease dividing, but they keep growing. As they are growing, the DNA
undergoes multiple rounds of replication making a large number of copies of each chromosome.
Such large number of copies are required to support the high level of secretory activity of these
enormous cells. The duplicated DNA strands remain attached to each other in perfect side-by-
side alignment, producing giant chromosomes with as many as 1024 times the number of DNA
strands of normal chromosomes.

Normally for a chromosome to become visible under a microscope, the cell has to enter mitotic
phase, where, the DNA undergoes a great level of condensation and become short and thick.
While in the case of the giant polytene chromosome, it is so thick (as a large number of DNA
molecules of the same chromosome type are stacked together from end to end) that, it is visible
under a microscope, in an interphase cell, without any condensation.

These unusual polytene chromosomes (so called, because they are not a single DNA molecule
but thousands of DNA stacked together), are rich in visual detail, displaying about 5000 bands
when stained and examined microscopically. The banding pattern is essentially constant from
one individual to the other. The individual bands on the chromosome could be correlated with
specific genes. The relative positions of these genes on the giant chromosomes agreed with those
predicted on the basis of genetic maps prepared from recombination frequencies, thus providing
visual confirmation of the validity of the entire mapping procedure.
Certain regions on these chromosomes become “puffed out” at particular stages of development.
These chromosome puffs are sites where DNA is being transcribed at a very high level,
providing one of the best systems available for the direct visualization of gene expression.

Reagents

Preparation of siliconized glassware: Siliconized glassware is needed to prevent tissue from


sticking to glass surfaces The easiest way to siliconize slides or coverslips is to immerse them for
a few minutes in silicon oil and to wipe them clean with tissues. (Pipettes used for oil should be
rinsed with alcohol.)

Drosophila Ringer solution: 6.7 g NaCl in 1000 ml distilled water.

Permanent Staining solution: Dissolve 2.2 g Orcein in 100 ml boiling glacial acetic acid;
dilute 1 : 1 with distilled water and filter only immediately before use.

Fixative: 3 parts absolute ethanol plus 1 part glacial acetic acid.

Procedure

Isolation of salivary glands

1. Third instar larvae are taken from uncrowded cultures.


2. For the preparation of especially large salivary gland chromosomes use larvae grown at
low temperature (below 18°C).
3. The larvae are raised in bottles without paper and are removed with a brush when they
start to leave the medium and crawl up the walls of the bottle towards the end of the third
larval instar.
4. Larva is held with two needles or two fine forceps in a drop of ‘Drosophila Ringer
solution’ and the anterior part with the mouth hooks is pulled away with a sudden
movement.
5. The posterior two thirds of the larva can be severed with a razor blade and discarded.
6. Both the neural ganglion and the two salivary glands with the joint opaque fat bodies are
found in the tissues that are attached to the anterior third of the larva.
7. With a fine needle and forceps the tissues are teased out under intermediate magnification
(20-25x).
8. The neural ganglion and the attached imaginal discs are separated;
9. The imaginal discs need not be removed.
10. The salivary glands are gently pulled free of the fat bodies which are discarded.
11. The larger cells lie in the center and at the posterior end of the salivary gland which
contains approximately 100 to 200 cells.

Preparation of Polytene chromosomes

1. The dissected salivary glands from one larva are put into a drop of Aceto-Orcein staining
solution.
2. The preparation is covered with a coverslip and left for about 3 min. The duration of the
staining can be modified with subsequent preparations depending on the effectiveness of
the staining solution.
3. The staining is terminated by soaking up the stain under the coverslip with a strip of filter
paper.
4. The coverslip is now covered with a broad piece of filter paper and the preparation is
squashed so that the cells and nuclear membranes burst and the chromosome arms are
spread. The simplest way is application of thumb pressure or tapping with a suitable
instrument (e.g. wooden handle of a dissecting needle).
5. To avoid desiccation of the preparation it is sealed along the edges of the coverslip with
clear nail polish.
6. Analyze the polytene chromosomes under a compound microscope.
Diagram to be drawn or printed and pasted in the record

F = fat bodies, G = gut, N = neural ganglion, S = salivary glands.

To be printed and pasted

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