Polytene Chromosomes in Drosophila

The fly, Drosophila melanogaster, is a holometabolous insect with four main stages to its lifecycle: embryo, larva, pupa, adult. As a larva, the organism is primarily concerned with obtaining food for the rapid increase in size characteristic of this stage of development. During this time, the salivary glands must be large and well-developed so that a sufficient supply of salivary enzymes is available for digestion. The salivary glands of Drosophila and some other insects achieve their growth through an increase in cell mass and volume rather than an increase in the number of cells. After an initial population of salivary gland cells is established during early larval development, cell division ceases. While the cells increase in size, the nuclei grow too, as the chromosomes duplicate repeatedly without accompanying cell division. Although the exact reason for this unusual process is unknown, it is apparently a more efficient way of producing salivary enzymes need for rapid larval development.

Since the salivary cells themselves are not dividing, the nuclei are not undergoing mitosis. The chromosomes are in an extended interphase of the cell cycle and, as such, are stretched out to their full length. Because each chromosome actually consists of many strands, they are called polytene (“many threaded”) chromosomes. Because polytene chromosomes are extended and consist of so much DNA, they are easily visible under the light microscope.

A useful feature of these chromosomes is that they have a pattern of dark and light bands, like a bar code, which is unique for each chromosome. The dark bands represent regions where the DNA is most densely packed, and the light bands (interbands) are regions where the DNA is less densely packed. These bands provide visible landmarks that can be used to identify the location of a specific gene on the chromosome or the sites of chromosomal rearrangements. Both bands and interbands contain genes and when a gene is being actively transcribed, puffs appear in the chromosomal region containing that gene. The puffs represent areas where the DNA is unwound from the normal densely-packed state so that it is accessible to the transcriptional machinery.

The standard genetic map of the polytene chromosome divides the genome into 102 numbered bands. The X chromosome comprises bands1-20, the second chromosome, bands 21-60; the third chromosome, bands 61-100; and the fourth chromosome (by far the smallest), bands 101-102. Each of these bands is divided into six “letter” bands (A-F) and those are subdivided into up to 13 numbered divisions. The location of many genes is known to the resolution of a letter band, usually with an estimate of the number location (e.g., 42C7-9, 60A1-2). Although the polytene divisions don't contain a consistent length of DNA, on average a letter band contains about 300 kb of DNA and between 15 and 25 genes.

There are three objectives for this laboratory: (1) To isolate the salivary glands from Drosophila larvae. (2) To prepare stained smears (“squashes”) of banded polytene chromosomes. (3) To observe polytene chromosome puffing after the chromosomes have been heat shocked.

Procedures

The success of these experiments is determined to a large extent by the developmental stage of the larvae. If they are too small, their salivary glands will not be sufficiently developed to yield good chromosomes. As the larvae become larger and approach pupation, the salivary glands begin to degenerate and are no longer suitable for chromosome preparation. Choose the largest larva you can find that is still moving actively; do not choose a larva that is noticeably darker than the rest, as darkening of the cuticle is a sign that pupation is imminent. Each student should prepare his or her own slides of polytene chromosomes. This will increase the chance that successful preparations will obtained in the class.

A. Preparing Salivary Squashes 1. Place a drop of 0.7% saline on a clean microscope slide. Transfer an appropriate larva (see above) to the slide and place it on the stage of a dissecting microscope.

2. Using the enhanced detail afforded by the microscope, firmly grasp the posterior end of the larva with forceps and use a dissecting needle, or another pair of forceps to pierce through the head, just behind the darkly pigmented mouthparts. Using a continuous motion, pull the needle (and attached head) away from the body.

The salivary glands are recognized by the following features. — They are paired and each is identical in size and shape. — They have a glistening, translucent appearance. — Each gland should have an opaque fat body associated with it.

3. While continuing to observe under the dissecting microscope, separate the salivary glands from any extraneous material, such as the fat bodies or parts of the digestive tract. Be careful not to damage the salivary glands themselves.

4. Once cleaned, remove the used saline and debris from the slide. Add fresh saline and allow the glands to soak for ten minutes.

There are two ways to perform the next step. The first is preferred since you can prepare two slides from one salivary gland. However, it is sometimes difficult to transfer each delicate gland to a separate slide. If you have trouble transferring the gland to a slide without damaging it, use step 5b.

5a. Obtain two fresh slides and add two drops of aceto-orcein stain to the center of each. Carefully pick up one of the salivary glands and transfer it to the drop of stain on one slide; transfer the second gland to the stain on second slide.

5b. Using a laboratory tissue, blot the saline from the slide containing the salivary glands in saline. Be careful not to touch the glands with the tissue as they will stick and it will not be possible to recover them. Place two drops of aceto-orcein stain directly on the glands.

6. Place the slides in a petri dish containing a moist filter paper (this prevents the stain from evaporating during incubation). Incubate for fifteen minutes.

7. Carefully blot excess stain away from the salivary gland. Be careful not to touch the glands with the tissue as they will stick. Add one drop of fresh stain and incubate for two minutes. (If you made two separate slides, leave the second gland soaking in the petri dish while you process the first).

8. Apply a cover slip to the stained salivary gland and place a paper towel or folded laboratory tissue over the cover slip. Using steady, moderate pressure, press down on the cover slip in a vertical direction. Do not twist the cover slip or allow it to move laterally; this will shear the chromosomes.

9. Observe the salivary gland squash at high-dry magnification with a compound microscope. Make sure that the optics are properly adjusted for Köhler illumination. A good preparation will reveal elongated chromosomes with distinct banding patterns.

If your preparation is promising, look closely for evidence of chromosomal puffing. If you find a puff, observe the chromosome with the oil immersion lens. If the preparation is not acceptable, try additional preparations with the second salivary gland (that is soaking in stain) or with a new salivary gland prep. Your instructor may direct members of the class to particularly good squashes prepared by other students in the lab.

B. Observing Heat Shock-induced Chromosomal Puffing. 1. Using a new, live larva, prepare salivary glands as described in steps 1 though 3 above. You should make two slides. One is labeled RT (room temperature) and the other labeled HS (heat shock).

2. Add fresh saline to each gland and incubate each in a humid petri dish. Place the HS slide in a 37°C incubator; the RT slide is incubated on the laboratory bench. Incubate the slides for 40 minutes. Do not exceed 40 minutes incubation.

3. Following incubation, carefully blot excess saline from the glands and stain as described in steps 5 and 6.

4. Squash the salivary gland on each slide as described in step 8 above.

5. Observe the chromosomes as described in step 9 above. You may have to scan several fields in order to identify chromosomes that show puffing. Compare the number of puffs in the RT and HS slides.

Post-lab Assignment:

Points to Consider

Polytene chromosomes have been invaluable for the study of genetics. What are some of the benefits of using polytene chromosomes? (You may have to consult a genetics textbook).

Explain how salivary enzymes might be produced more efficiently from polytene chromosomes when compared to normal chromosomes.

Why does incubation of the chromosomes at 37°C induce puffing?

Are there other treatments that you can think of that might also induce puffing?