Experiment 06Structural CytogeneticsGiant Chromosomes
Temporary Preparation & Microscopic Examination of Giant Polytene Chromosomes from Dipteran Salivary Glands
Lab Duration: 3 Hours • Larval Dissection & Cytological Squash
🎯 Aim: To dissect paired salivary glands from third-instar larvae of Chironomus (midge) or Drosophila melanogaster, remove adherent fat bodies, prepare a temporary aceto-orcein or aceto-carmine squash mount, and examine the ultrastructural banding patterns, chromocenter, and transcriptionally active Balbiani rings (chromosome puffs) of giant polytene chromosomes under brightfield microscopy.
🔬 Principle & Molecular Cytology
Polytene chromosomes were first discovered by Édouard-Gérard Balbiani in 1881 in the salivary glands of Chironomus larvae. They are giant interphase chromosomes formed through repeated cycles of DNA replication without cell division or chromosome segregation—a specialized cell cycle variant termed endoreduplication (or endomitosis).
In salivary gland cells of Drosophila, chromosomal DNA undergoes 9 to 10 successive rounds of replication ($2^{10} = 1024$ sister chromatid strands per chromosome). Because somatic homologous chromosomes undergo permanent, tight lateral pairing (somatic synapsis), maternal and paternal homologs align in perfect register, producing a cable-like multistranded nucleoprotein fiber containing over $1000$ to $2000$ parallel chromonemata.
- Chromomeres & Dark Bands: Condensed chromatin coils containing approximately $95%$ of chromosomal DNA. They stain deeply with basic nuclear dyes (aceto-orcein, Feulgen, Giemsa). Over $5000$ distinct bands have been mapped in Drosophila.
- Interbands: Stretched, less tightly coiled euchromatic segments representing only $5%$ of total DNA, staining weakly.
- Balbiani Rings (Puffs): Localized decondensation and lateral looping of chromatin fibers at specific gene loci. Puffs represent sites of intense transcription by RNA Polymerase II, synthesizing enormous quantities of messenger RNA coding for salivary glue proteins required for pupal adhesion. Puffs expand and contract dynamically in response to the insect steroid molting hormone 20-hydroxyecdysone.
- Chromocenter: In Drosophila, the centromeric heterochromatin of all 4 pairs of chromosomes (X, 2, 3, and small 4) coalesces into a single dark, amorphous central heterochromatic mass from which five long polytene chromosome arms radiate outward (X, 2L, 2R, 3L, 3R) along with one tiny stump (chromosome 4).
📋 Step-by-Step Dissection & Squash Protocol
- Larval Selection: Select large, plump, well-fed third-instar larvae that have crawled out of the culture food onto the dry glass walls of the culture vial prior to pupation. Larvae cultured at lower temperatures ($18^circtext{C}$) possess substantially larger salivary glands and higher polyteny levels.
- Dissection Setup: Place the larva in a small drop of physiological saline ($0.7%,text{NaCl}$) on a clean microscope slide under a stereomicroscope with transmitted illumination.
- Gland Extraction: Hold the posterior third of the larva firmly with one dissection needle. Place a second needle across the anterior prothoracic region just behind the black mouth hooks. Gently pull the two needles in opposite directions. The head, mouth hooks, and alimentary canal will pull free, drawing out the paired, transparent, glistening, sausage-shaped salivary glands.
- Fat Body Trimming (Critical Step): The salivary glands have an opaque, chalky-white fat body attached along their lateral margins. Using micro-dissecting needles, carefully scrape away and discard all white fat body tissue. If fat remains, it will cloud the squash and prevent chromosome spreading.
- Fixation & Staining: Immediately transfer the cleaned glands into a drop of $2%$ Aceto-Orcein or $1%$ Aceto-Carmine stain on a clean slide. Allow the tissue to fix and stain for 10 to 15 minutes. (Acetic acid fixes nucleoproteins while orcein/carmine binds basic chromatin).
- Squashing Technique: Carefully place a clean coverslip over the stained glands. Invert a piece of clean bibulous filter paper over the slide. Apply firm, direct downward thumb pressure on the coverslip without allowing any lateral twisting or shearing motion. This flattens the giant nuclei, ruptures the nuclear envelope, and spreads the polytene arms into a single optical focal plane.
- Microscopic Examination: Seal edges of coverslip with clear nail polish. Examine under $10times$ to locate polytene chromosome spreads, then switch to $40times$ and $100times$ oil immersion to observe detailed band-interband sequences and Balbiani ring puffs.
