(a) What is recombinant DNA technology? Describe the different vectors used in DNA cloning. (20 marks) (b) Describe the…

(a) Recombinant DNA technology and cloning vectors (20 marks)

Recombinant DNA technology. It is the set of techniques by which DNA from different sources is cut and joined in the test tube and introduced into a host cell, where it replicates and may express (Cohen and Boyer, 1973).

Essential steps.

• Isolate the gene of interest and the vector DNA.
• Cut both with the same restriction endonuclease (EcoRI, for example, leaves sticky ends).
• Join them with DNA ligase to make the recombinant molecule.
• Introduce it into a host by transformation (CaCl₂ and heat shock, or electroporation), transfection, microinjection, gene gun or Agrobacterium.
• Select recombinants by antibiotic resistance, insertional inactivation or blue–white screening, and grow them for expression and the product.

Vectors. A vector is a DNA molecule that carries foreign DNA into a host. It needs an origin of replication, a selectable marker and unique cloning sites. The main types are:

• Plasmids: small circular DNA of bacteria, taking inserts up to about 10 kb, such as pBR322 (ampicillin and tetracycline resistance) and the high-copy pUC18/19 with lacZ for blue–white screening.
• Bacteriophage λ vectors: insertion and replacement types, taking about 20–23 kb, and packaged in vitro into phage particles, so infection is efficient.
• Cosmids: plasmids with the cos site of λ, taking up to 45 kb.
• Phagemids: plasmid and M13 features, giving single-stranded DNA (pBluescript).
• Artificial chromosomes: BACs (from the F plasmid, 100–300 kb, used in genome sequencing), PACs (from phage P1, similar size) and YACs (yeast, with centromere, telomeres and ARS, 200–2,000 kb).
• Shuttle and expression vectors: replicate in two hosts (E. coli and yeast), or, like pET, carry strong promoters for protein production.
• Ti plasmid of Agrobacterium for plants.
• Viral vectors (retrovirus, adenovirus, AAV, baculovirus for insect cells) for animal cells and gene therapy.

The choice depends on insert size, the host and the purpose.

(b) Applications of cladistics in modern taxonomy (15 marks)

Cladistics (Hennig) classifies organisms by shared derived characters (synapomorphies) into nested branching groups, shown as a cladogram, and recognises only monophyletic groups (clades) that contain an ancestor and all its descendants. Its uses are as follows.

• Reconstruction of phylogeny from morphological and molecular characters, by maximum parsimony, maximum likelihood or Bayesian methods, using an outgroup to root the tree.
• Monophyly-based classification: birds are placed within the theropod dinosaurs, so "Reptilia" without birds is paraphyletic, and whales fall within the even-toed ungulates (Cetartiodactyla), being sister to hippopotamuses.
• Species delimitation: the phylogenetic species concept identifies cryptic species, as in many frogs and insects.
• Divergence dating: the tree is calibrated with the fossil record and a molecular clock to date splits.
• Conservation: phylogenetic diversity helps to prioritise evolutionarily distinct lineages, as in the EDGE programme for the tuatara and aye-aye.
• Biogeography and comparative biology: they test vicariance and trace the evolution of characters, such as flight, or C₄ photosynthesis in plants.
• Epidemiology: phylogenies of HIV and SARS-CoV-2 track the origin and spread of outbreaks.

(c) Mitosis and meiosis (15 marks)

• Site and cell type: mitosis occurs in somatic cells and in gonial cells, and meiosis in germ-line cells (gonads, or spore mother cells in plants).
• Divisions and products: mitosis is one division giving 2 daughter cells; meiosis is two divisions (I and II) giving 4 cells.
• Chromosome number: mitosis keeps it (2n → 2n); meiosis halves it (2n → n).
• DNA replication: one S phase before mitosis, and one before meiosis I, none between I and II.
• Prophase: short in mitosis; long in meiosis I, with five stages (leptotene, zygotene, pachytene, diplotene and diakinesis).
• Homologues: they do not pair in mitosis; in meiosis I they pair (synapsis) into bivalents.
• Chiasmata and crossing over: absent in mitosis; present in pachytene and seen as chiasmata in diplotene.
• Metaphase and anaphase: in mitosis the chromosomes line up singly and the centromeres split, so sister chromatids separate. In anaphase I homologous chromosomes separate and centromeres do not split, and in anaphase II the centromeres split and sister chromatids separate.
• Genetic result: mitosis gives genetically identical cells; meiosis gives genetically different cells by crossing over and independent assortment.
• Significance: mitosis is for growth, repair and asexual reproduction; meiosis makes gametes or spores, keeps the chromosome number constant across generations and creates variation.