(a) Frame Shift mutation (10 marks) (b) Lampbrush chromosome and its significance (10 marks) (c) Reporter genes (10 marks) (d)…

(a) Frameshift mutation (10 marks)

A frameshift mutation is the insertion or deletion of nucleotides, in a number not divisible by three, in a coding sequence. Since the code is read in non-overlapping triplets, the reading frame shifts and every codon downstream is altered. For example, AUG AAA GCU CCU becomes AUG AAG CUC CU when the fourth base is deleted.

Consequences: all later amino acids change and a premature stop codon is usually met, giving a truncated, non-functional protein, so the effect is generally severe; the mRNA is often destroyed by nonsense-mediated decay.

Causes: intercalating agents such as acridine dyes (proflavine, acridine orange), and polymerase slippage in repeats. Examples: the 32-bp deletion in CCR5 (CCR5-delta32) and the 4-bp insertion in HEXA that causes Tay-Sachs disease.

Crick, Brenner and co-workers used T4 rII mutants in 1961 to show that adding or removing one or two bases inactivates the gene but three restore the frame, proving that the code is a triplet.

(b) Lampbrush chromosome and its significance (10 marks)

Lampbrush chromosomes are giant chromosomes seen in the diplotene stage of meiotic prophase I in the oocytes of amphibians (Triturus, Xenopus) and also of reptiles, birds, fish and insects. They were first described by Flemming (1882) and named by Rückert (1892), and are 0.5 to 1 mm long.

Structure:

• Bivalents are held together by chiasmata.
• Each chromosome has a central axis of condensed chromomeres carrying DNA.
• Pairs of lateral loops, one for each sister chromatid, project from the chromomeres like the bristles of a lamp brush, thin at one end and thicker at the other.
• The loops carry an RNA-protein matrix of nascent transcripts and are active in transcription.

Significance:

• They are sites of intense transcription during oogenesis, storing RNA for early embryonic development.
• They are a model for gene expression, transcription units and RNA processing.
• They help in gene mapping by in situ hybridisation and in the study of chromosome structure and chiasmata.

(c) Reporter genes (10 marks)

A reporter gene is a gene whose product is easily detected, and it is fused to a promoter or another gene to monitor gene expression, promoter activity and the success of transformation.

Properties of a good reporter: it is absent from the host or has very low background activity; its product is quickly, sensitively and quantitatively assayed; and it is non-toxic, stable and small.

Examples:

• lacZ (beta-galactosidase), which turns X-gal blue.
• uidA (GUS, beta-glucuronidase), which turns X-Gluc blue, widely used in plants; the assay destroys the tissue.
• Green fluorescent protein (GFP) from the jellyfish Aequorea victoria, which fluoresces under blue or UV light with no substrate, allows live imaging and localises proteins fused to it.
• Luciferase (firefly), which emits light with luciferin and ATP, very sensitive and real-time.

Uses: studying tissue-specific promoters, protein localisation and screening of transformants. A reporter differs from a selectable marker such as nptII (kanamycin resistance), which lets only transformed cells survive.

(d) Cell signalling and cell receptors (10 marks)

Cell signalling is the communication between cells and within them, in which a signal (ligand) is received by a receptor, transduced and produces a response.

Types: endocrine (hormones carried through the body), paracrine (nearby cells), autocrine (the same cell) and juxtacrine, contact-dependent (membrane-bound ligands, plasmodesmata, gap junctions). Signals include hormones, growth factors, neurotransmitters, light, auxin, ethylene and pathogen elicitors.

Transduction uses second messengers (cAMP, Ca²⁺, inositol trisphosphate, diacylglycerol) and phosphorylation cascades (MAP kinases), ending in altered gene expression, and is then switched off.

Receptors:

• Cell-surface receptors for signals that cannot cross the membrane: G-protein-coupled (seven-transmembrane) receptors, enzyme-linked receptors such as receptor tyrosine kinases, receptor-like kinases in plants (BRI1, FLS2, CLV1) and ligand-gated ion channels.
• Intracellular receptors for lipid-soluble signals: steroid hormone receptors; in plants, phytochrome for red and far-red light and the receptors of auxin (TIR1), gibberellin (GID1) and abscisic acid (PYR/PYL).

(e) DNA fingerprinting (10 marks)

DNA fingerprinting, developed by Alec Jeffreys in 1984, identifies individuals from their DNA. It rests on hypervariable repeats, minisatellites or VNTRs (repeats of 10 to 100 bp) and microsatellites or STRs (repeats of 2 to 6 bp), whose repeat number differs among people, so the pattern is unique except in identical twins.

Method:

1. Isolate DNA from blood, hair, semen or tissue.
2. Cut with a restriction enzyme (HaeIII, HinfI) and separate the fragments by agarose gel electrophoresis.
3. Transfer to a membrane (Southern blotting) and hybridise with a labelled VNTR probe.
4. Detect by autoradiography to give a barcode-like pattern.

Modern practice amplifies STR loci by PCR with fluorescent primers and reads them by capillary electrophoresis, needing only a tiny sample.

Applications: forensic cases, paternity disputes, identification of disaster victims, wildlife forensics, and, in plants, varietal identification and diversity studies. In India it was pioneered at CCMB, Hyderabad, by Lalji Singh.