Polytene Chromosome Preparation from Drosophila or Chironomus Salivary Glands
Polytene chromosomes are giant, multi-stranded chromosomes discovered by E.G. Balbiani in 1881 in Dipteran dipteran salivary gland nuclei. They are produced by specialized cell cycles termed endoreduplication (endocycles), in which repeated rounds of DNA replication ($S$-phase) proceed without intervening mitotic spindle assembly, nuclear envelope breakdown, or cytokinesis ($M$-phase).
In Drosophila melanogaster, larval salivary gland cells undergo 9–10 consecutive endocycles, producing $2^{10} = 1024$ tightly synapsed chromatid strands per homolog. Permanent somatic pairing fuses homologous maternal and paternal chromosomes, creating a unified cable-like giant structure containing up to 2048 chromatids, approximately 100 to 200 times larger than standard metaphase chromosomes.
Along the chromosome length, differential chromatin condensation generates alternating transverse bands (chromomeres) and interbands. Bands are rich in tightly packed, transcriptionally silent DNA ($95%$ of nuclear DNA), staining deeply with basic dyes like aceto-orcein. Interbands are less condensed and represent active or accessible DNA. At active transcription loci, chromatin fibers unwind and loop out into localized swellings called puffs or giant Balbiani rings, where active RNA synthesis and ecdysone-regulated transcription occur.
All centromeric heterochromatic regions aggregate into an amorphous, deeply staining central mass termed the chromocenter, from which five long arms (X, 2L, 2R, 3L, 3R) and one tiny dot-like arm (chromosome 4) radiate outward.
- 2% Aceto-Orcein Stain: Dissolve 2.0 g certified orcein powder in 100 mL boiling 45% glacial acetic acid; reflux 30 min, cool, filter through Whatman No. 1.
- 1N Hydrochloric Acid (1N HCl): For tissue softening/maceration.
- Insect Ringer’s Solution: $0.75% text{NaCl}, 0.035% text{KCl}, 0.021% text{CaCl}_2$ in distilled water (pH 7.2).
- 45% Glacial acetic acid & clear fingernail polish (for ringing coverslip).
- Healthy culture of Drosophila melanogaster maintained on cornmeal-yeast-agar medium at 22–25°C
- Stereoscopic zoom dissection microscope (10×–40×) with top illumination
- Dumoxel No. 5 watchmaker’s tweezers (two pairs) & tungsten needles
- Standard compound microscope (10×, 40×, 100× oil)
- Larval Selection: Select the largest, well-fed 3rd-instar wandering larvae crawling upward along the culture bottle glass wall (just prior to pupation). These larvae possess fully developed, endoreduplicated salivary glands.
- Dissection in Ringer’s: Place a larva in a drop of insect Ringer’s solution on a clean glass slide under a dissection stereomicroscope. Position one pair of fine forceps firmly across the posterior third of the larval body. Place the second pair of forceps at the anterior mouth hooks (black cephalopharyngeal skeleton).
- Gland Extraction: Pull the two forceps steadily and gently in opposite directions. The head will detach, pulling out the alimentary canal and a pair of elongated, transparent, glistening, sausage-shaped salivary glands.
- Fat Body Trimming: Salivary glands are flanked by opaque white adipose tissue (fat bodies). Using fine micro-dissection needles, cleanly tease away and discard all fat bodies; failure to remove fat results in cloudy, greasy squashes that obscure chromosome bands.
- Fixation & Staining: Blot away excess Ringer’s. Add a drop of 1N HCl for 60 seconds to soften cell membranes. Blot acid, then immediately add 2 drops of 2% aceto-orcein. Allow the glands to stain in the dark for 10–15 minutes (cover with an inverted petri dish to prevent evaporation).
- Squash & Spreading: Lower a clean coverslip onto the stained glands. Invert a fold of filter paper over the slide. Apply strong, steady, vertical thumb pressure without any lateral sliding. Check spread under 10×; if chromosomes are well-spread, seal coverslip edges with clear nail polish.
| Chromosome Arm | Relative Length | Cytogenetic Diagnostic Markers |
|---|---|---|
| Chromocenter | Central Hub | Dark, amorphous mass formed by fused centromeric heterochromatin of all chromosomes. |
| X Chromosome | Medium Long | In males ($XY$), stains paler and appears thinner due to dosage compensation; distinct flared tip. |
| Chromosome 2L | Long Arm | Characteristic blunt tip; prominent constrictions and distinct double bands at region 21. |
| Chromosome 2R | Long Arm | Tapered tip; prominent terminal Balbiani ring / puff locus at late 3rd instar. |
| Chromosome 3L | Long Arm | Squared, duck-bill shaped distal tip; distinctive dark triplet band at region 61. |
| Chromosome 3R | Longest Arm | Prominent ecdysone-responsive puffing sites (e.g. 74EF, 75B); rounded club-shaped tip. |
| Chromosome 4 | Very Short | Tiny dot-like chromosome radiating directly from the chromocenter; $approx 4–5$ visible bands. |
- Never use pupae or young 1st/2nd-instar larvae; polyteny only reaches maximal 1024-strand polyteny in late wandering 3rd-instar larvae.
- Completely strip the white fatty lobes from the salivary glands: fat droplets create refractive optical artifacts that completely mask banding.
- Avoid drying of the stain during the 15-minute incubation period; if acetic acid evaporates, orcein precipitates as dark, needle-like dye crystals.
