Polytene Chromosome

Drosophila is an excellent genetic research organism because fruit flies:
• have a short generation time (important for research spanning a number of generations)
• are small and easy to keep in a laboratory
• produce reasonably good numbers of offspring
• have a number of easy to see inheritable characteristics
• have a chromosome number of 8 (4 pairs of chromosomes)

Insects typically have multiple life cycle stages, including egg, larva, pupa and adult. The fruit fly is typical of those insects whose larval stage is the assimilative stage. Larvae may pass through several stages before they pupate. After pupating for the requisite time, adults hatch out and find mates. Adults generally are short-lived.

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 are polytene.

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 

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.

Polytene chromosomes are usually found in the larvae, where it is believed these many-replicated chromosomes allow for much faster larval growth than if the cells remained diploid. Simply because each cell now has many copies of each gene, it can transcribe at a much higher rate than with only two copies in diploid cells. 

The polytene chromosomes at the right are from the salivary glands of the fruit fly Drosophila melanogaster. the bands on each chromosome are like a road map, unique to each chromosome and well defined enough to allow high resolution mapping of each chromosome.

Procedure

1. Prepare a clean slide with 2-3 drops of PBS (Phosphate Buffer Saline) and put the slide (without a cover slip) on a stereoscopic (dissecting) microscope.
2. Select a large Drosophila melanogaster larva and place it on the slide.
3. While looking through the microscope use probes or forceps to grasp the larva by its
   midsection just behind its jaws.
4. Gently stretch the larva by pulling on it until its head separates from the rest of its body.
5. Look for the salivary glands in the head section. The glands are very small, fairly
   transparent, usually paired and have dark fat particles attached.

6. When you have located the salivary glands, separate them from the rest of the fruit fly
tissues. Once you are certain that you have successfully done this, you may dispose of
the rest of the larva appropriately. Keep the salivary glands moist with PBS at all
times. Do not let it dry.

7. Add 2-3 drops of aceto-orcein stain to your Drosophila salivary glands and keep it for 5-10 mins.

8. After the stain has set, get two paper tissue and place your slide on one of them. Put a coverslip on the slide (on top of the salivary glands). Fold the second tissue and place it on top of the coverslip.

9. Place your thumb on the over the coverslip and press down slowly and firmly. Use sufficient pressure but do not allow the coverslip or slide to slip or move.
12. Examine your stained, squashed salivary glands using the medium power objective lens. Look for nuclei and chromosomes. After you have located chromosomes, use the high power objective lens to see details of the chromosomes.