Polytene Chromosomes in the Salivary Glands of Drosophila

Renren Barroga, Ma. Rica Paulene Marquez

Department of Biology - University of the Philippines Baguio
ABSTRACT
Polytene chromosomes are result of repeated DNA synthesis without division into daughter
chromosomes. This experiment aims to aims to orient students with the proper technique in isolation
of polytene chromosomes from D. melanogaster salivary glands and to explain basic concepts about
the chromosome. Polytene chromosome was observed in this experiment by extracting salivary
glands from third instars of Drosophila melanogaster. Salivary glands were stained with aceto-orcein
and observed under a light microscope.
INTRODUCTION
In certain cells and tissues of some insects and
amphibians, chromosomal structures can be observed
that relate to their function. Polytene (many threads)
chromosomes are formed as a result of repeated DNA
synthesis without cell division, have a distinct pattern of
chromosome banding readily visible under the light
microscope. These chromosomes are first observed by
E.G. Balbiani in cells of salivary glands of Drosophila
melanogaster and Chironomus. (Passarger, 2007)
These polytene chromosomes are a result of
ten cycles of replication without division into daughter
chromosomes. Thus, there are about 1024 (2 10)
identical chromatid strands, which lie strictly side by
side.
(Passarge,
2007)
Generally,
polytene
chromosomes aare much larger than metaphase
chromosomes both because of the multiple chromatids
and because of lower compaction (Goldstein and
Fyrberg, 1994). The Drosophila genome contains about
5000 bands (Passarge, 2007).
This experiment aims to aims to orient students
with the proper technique in isolation of polytene
chromosomes from D. melanogaster salivary glands and
to explain basic concepts about the chromosome.

METHODOLOGY
Healthy-looking third instar larvae were
obtained from stock of wild fruitflies. The food around
the larvae was rinsed off in a petri dish. The larvae were
then transferred to moistened glass slide after it has
been rinsed. Using two dissecting needles, the mouth
hooks were pulled together with the attached
structures from the larval body. The salivary glands
were then separated from the other tissues. Once the
unwanted tissues were removed from the slide, the

salivary glands were stained with aceto-orcein for ten

minutes. A cover slip was then placed in top of the
specimen. Enough pressure was then applied over the
specimen to crush the cells and spread the
chromosomes. The excess stain was blotted off. After
which, the specimen was observed under a light
microscope. Two slides of clearly discernable polytene
chromosomes were prepared in this experiment.

RESULTS AND DISCUSSION

Drosophila melanogaster is a holometabolous
insect with four main stages to its lifecycle: embryo,
larva, pupa, and adult. As a larva, the organism is
primarily concerned with obtaining food for the rapid
increase in size. During this time, salivary glands must
be large and well developed to sufficiently supply
salivary enzymes which will be used for food digestion.
These salivary glands achieve their growth through an
increase in cell mass and volume rather than an
increase in cell number (Clark, 2015). After an initial
population of salivary gland cells is established during
early larval development, cell division ceases (Clark,
2015).
Since the salivary cells are not dividing, the
nuclei are not undergoing mitosis. Thus, the
chromosomes are in an extended interphase of the cell
cycle and are stretched out to their full length. The
exact reason for this unusual process, increase in cell
mass rather than cell number, is still unknown;
however, it is apparently a more efficient way of
producing salivary enzymes need for rapid larval
development
(Clark, 2015). Because polytene
chromosomes are extended and consist of so much
DNA, they are easily visible under the light microscope.
Different methods were carefully done to prepare the
polytene chromosome. The success of the experiment is

determined to a large extent by the developmental

stage of the larvae (Clark, 2015). If they are too small,
their salivary glands will not be sufficiently developed to
yield a good chromosome. If they are too large and
approaching pupation, the salivary glands begin to
degenerate and are no longer suitable for chromosome
preparation. Larvae that are approaching pupation are
visibly darker than the rest due to the darkening of the
cuticle (Clark, 2015).

mitosis, thus, salivary cells increase in cell mass rather

than in cell number. These chromosomes are called
polytene chromosomes. A pair of salivary glands was
isolated from appropriate larvae (third instar larvae).
From these salivary glands, the polytene with distinct
banding was observed.
Problems aroused during the exercise. The
aceto orcein used was either diluted or expired which
caused the incomplete staining of the chromosomes,
thus, banding is not distinctly visible. It was also
observed applying pressure could squash the salivary
glands, thus, making them flattened and distorted.

QUESTIONS FOR RESEARCH

Figure 1 Salivary Glands

After obtaining the appropriate larva, the
salivary glands were obtained by piercing the head and
pulling it away from the body (Figure 1). The salivary
glands are paired and each is identical in size and shape,
and has a glistening, translucent appearance (Cai, et. al.,
2010). The salivary glands were then stained using
aceto orcein. Aceto orcein was used to label
chromosomal loci (Cai, et. al., 2010). A cover slip was
then placed in top of the specimen. Enough pressure
was then applied over the specimen. This pressure
forces the salivary gland cells to burst open and the
chromosomes to spread out (Cai, et. al., 2010). Figure 2
shows the prepared polytene chromosome.

CONCLUSION
Salivary
chromosomes
of
Drosophila
melanogaster in the larval stage does not undergo

the

Chromosomes are coiled strands of DNA that

appear inside the nucleus during cell division. There are
various
ways
of
classifying
chromosomes.
Chromosomes can be classified according to position of
centromere: acrocentric chromosomes (those with
centromere is present near one end), metacentric
(centromere is situated at the center), submetacentric
(centromere is situated between the midpoint and at
one end of the chromosome), and telocentric
(centromere is situated at the end having only one
arm). Another way of classifying chromosomes is by the
Denner-London System classification, according to this
system chromosomes are classified in different groups
according to their length. And lastly, chromosomes can
also be classified using the Paris conference (1972)
classification, wherein chromosomes are classified
based on banding pattern. This system of classification
provides more accuracy to the identification of parts of
each chromosomes.

Figure 2 Polytene Chromosomes

What are chromosomes? Enumerate

criteria used in classifying chromosomes.

Explain the different methods of staining

chromosomes.

Chromosome banding methods are either

based on staining chromosomes with a dye or on
assaying for a particular function. The most common
methods of dye based chromosome banding are G(Giemsa), R-(reverse), C-(centromere) and Q(quinacrine) banding. Bands that show strong staining
are referred to as positive bands; weakly staining bands
are negative bands. However the staining patterns are
not black and white, different bands stain to different
intensities (Francke, 1994). G-positive bands are usually
just called G-bands and likewise for Rpositive (R-) bands.

Positive C-bands contain constitutive heterochromatin.

Q-bands are considered equivalent to Gbands.
The most widely used function-based banding
method is replication banding and is based on the fact
that different bands replicate theirDNAat different
times during S phase of the cell cycle. Generally, R-band
DNA is replicated earlier than G-bands (Dutrillaux et al.,
1976). G-bands also correspond to the condensed
chromomeres of meiotic chromosomes and R-bands to
the interchromomeric regions.

Give the importance of chromosome number.

Chromosome number is important in the

context of species perpetuation. Parents should have
the same amount of chromosomes to pass on to their
offspring. These chromosomes must match up
(successful homologous pairing) in order for the
offspring to become fertile. Therefore, if a chromosome
number was not maintained in a species, the species
would not be able to mate.
Differing chromosome numbers alter the
genetic code of the organism. Any change in an
organism's genetic code is called a mutation. All
mutations are random. Although a small percentage can
be potentially beneficial, many of them are deleterious
(meaning they damage or cause harm to the organism).
Occasionally, the correct numbers of chromosomes are
not passed onto offspring for various reasons. One
well-known disease caused by a chromosomal disorder
is Down Syndrome. This disorder is caused by an extra
copy of chromosome 21.

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