(a) Describe the steps of constructing a recombinant DNA and its cloning. (20 marks) (b) Discuss the major morphological…

(a) Construction of recombinant DNA and its cloning

Recombinant DNA (rDNA) technology joins a DNA fragment of interest to a self-replicating vector and multiplies it in a host cell; the first recombinant plasmid was made by Cohen and Boyer (1973). The steps are as follows.

• Isolation of DNA: the source DNA carrying the gene of interest, and the vector DNA, are extracted by cell lysis, removal of proteins and RNA with proteases and RNases, and ethanol precipitation. Alternatively, the gene is obtained as complementary DNA (cDNA) made from mRNA by reverse transcriptase (needed for eukaryotic genes with introns), or amplified by PCR.
• Choice of vector: vectors need an origin of replication, a selectable marker and unique restriction sites (a multiple cloning site). Plasmids such as pBR322 and pUC19 carry inserts up to about 10 kb; bacteriophage λ vectors about 20 kb; cosmids 35–45 kb; bacterial artificial chromosomes (BACs) 100–300 kb; and yeast artificial chromosomes (YACs) up to about 1 Mb.
• Cutting with restriction endonucleases: the source and vector DNA are cut with the same enzyme, for example EcoRI (recognising GAATTC), producing complementary sticky ends; enzymes such as SmaI give blunt ends.
• Ligation: the fragment and the opened vector are mixed, the sticky ends base-pair, and DNA ligase seals the phosphodiester backbone, producing a recombinant molecule. Dephosphorylating the vector with alkaline phosphatase reduces self-ligation.
• Transformation: the recombinant DNA is introduced into a host, usually Escherichia coli made competent with calcium chloride and heat shock, or by electroporation; yeast (Saccharomyces cerevisiae) or animal cells are used when eukaryotic processing is needed. Microinjection, gene guns and viral vectors are other delivery methods.
• Selection of transformants: an antibiotic-resistance marker (such as ampR) lets only cells that took up a vector grow on antibiotic medium.
• Screening for recombinants: insertional inactivation separates recombinants from empty vectors. In pBR322 an insert in the tetracycline-resistance gene makes cells ampicillin-resistant but tetracycline-sensitive (replica plating). In pUC vectors an insert in lacZα abolishes β-galactosidase, so recombinant colonies are white and non-recombinants blue on X-gal with IPTG. Colony hybridisation with a labelled probe, colony PCR and sequencing confirm the correct clone.
• Cloning and expression: the selected colony is grown in culture, multiplying the recombinant DNA along with the cells. With an expression vector carrying a suitable promoter, the protein can be produced on a large scale, as for human insulin in E. coli (Genentech, approved 1982), hepatitis B surface antigen in yeast, and Bt toxin genes in crops.

(b) Major morphological modifications during horse evolution

The horse lineage, from Eohippus (Hyracotherium) of the early Eocene about 55 million years ago to Equus, is a classic, well-documented fossil series, mostly from North America. G.G. Simpson (1951) showed that it is a branching bush with many side branches and extinctions rather than a straight-line (orthogenetic) progression. The main driver was the spread of dry, open grasslands in the Miocene, which favoured fast running and grazing on abrasive grasses.

• Body size: from the fox-sized Eohippus (about 25–50 cm at the shoulder) to the large modern horse (about 1.6 m), although some lineages became smaller.
• Limbs and digits: Eohippus had four toes on the forefoot and three on the hindfoot, each ending in a small hoof, and walked on pads. Mesohippus (Oligocene) was three-toed on all feet, with the middle toe larger. In Merychippus (Miocene) the side toes no longer touched the ground in normal stance, and weight rested on the enlarged third digit. Pliohippus (Pliocene) was essentially one-toed, and in Equus the third digit alone bears the weight, ending in a single hoof, while digits II and IV persist only as splint bones. The limbs lengthened, especially the metapodials, the ulna and fibula were reduced and fused to the radius and tibia, and joints became hinge-like, restricting movement to the fore-and-aft plane for efficient running (unguligrade posture).
• Dentition: browsing Eohippus had low-crowned (brachyodont) teeth with low cusps. From Merychippus onward the cheek teeth became high-crowned (hypsodont), with complex enamel folds, cement filling and continued eruption to resist wear from silica-rich grasses; the premolars became molar-like (molarisation), forming a continuous grinding battery separated from the incisors by a diastema.
• Skull and brain: the face lengthened in front of the eyes to house the tall teeth and deep jaws, the orbit moved back and was enclosed by a post-orbital bar, and the brain enlarged, especially the cerebral hemispheres with more complex folding.

In India the Siwalik deposits have yielded three-toed hipparionines (Hipparion theobaldi) and the Pleistocene Equus sivalensis, showing that horses dispersed from North America into the Old World several times.

(c) Salient features of zoological nomenclature

Zoological nomenclature is the system of scientific names for animals, governed by the International Code of Zoological Nomenclature (4th edition, 1999, in force from 1 January 2000) and administered by the International Commission on Zoological Nomenclature (ICZN). Its main features are:

• Independence: zoological names are independent of botanical and bacterial names, so the same generic name may be used in both kingdoms.
• Binominal nomenclature: each species has a two-part Latin or Latinised name, the genus capitalised and the specific name in lower case, both italicised (e.g., Panthera tigris). A third name denotes a subspecies (e.g., Panthera tigris tigris).
• Starting point: zoological nomenclature starts from 1 January 1758, the date assigned to the 10th edition of Linnaeus's Systema Naturae (and Clerck's Aranei Svecici).
• Availability: a name must be published in a work meeting the Code's criteria, with a description or definition; electronic works must be registered in ZooBank (since 2012).
• Principle of priority: the valid name of a taxon is the oldest available name. Later names for the same taxon are junior synonyms and are invalid; the Commission may conserve a well-established name under its plenary powers.
• Principle of homonymy: identical names for different taxa in the same group are homonyms; the junior homonym must be replaced by a new name (nomen novum).
• Principle of typification: every nominal taxon has a name-bearing type: a holotype, syntypes, lectotype or neotype for a species; a type species for a genus; and a type genus for a family. Types fix the application of names when taxa are split or revised.
• Principle of coordination: names within the family group, genus group or species group are coordinate; establishing a name at one rank establishes it at the others in that group with the same author and date.
• Author and date: the author's name follows the scientific name without punctuation, and a comma separates it from the year (e.g., Homo sapiens Linnaeus, 1758). The author and date are placed in parentheses when the species has been moved to a different genus (e.g., Naja naja (Linnaeus, 1758)).
• Family-group endings: superfamily -oidea, family -idae (e.g., Felidae), subfamily -inae and tribe -ini, formed from the stem of the type genus. The Code does not regulate names above the family group.
• Tautonymy and gender agreement: tautonyms (identical genus and species names, e.g., Naja naja, Bison bison) are permitted in zoology, unlike botany; an adjectival specific name must agree in gender with the genus.