Biotechnology & Molecular Biology BBO6T14

Syllabus topics (RTMNU)

• Biotechnology: Definition and scope of biotechnology
• Plant tissue culture: Cellular totipotency, Explant, Aseptic culture, Micropropagation, De-differentiation, Re-differentiation and Morphogenesis
• Plant tissue culture laboratory: Plant tissue culture cabinets, preparation room, Oven, Autoclave, Laminar Air Flow, pH meter, glassware
• Media: Basal medium; Preparation and composition of Murashige T. and F. Skoog, 1962 (M.S.)
• Methods of sterilization: Physical methods (dry heat, moist heat (autoclave), Filter sterilization and UV)
• Chemical methods: Ethyl alcohol, Calcium hypochlorite, Sodium hypochlorite, Mercuric chloride, H2O2 and antibiotics
• Whole plant culture: Embryo culture and its importance
• Organ culture: Anther culture, Shoot tip culture and their importance
• Callus culture and its importance
• Protoplast culture and its importance
• Synthetic seed production, advantages and disadvantages of synthetic seeds
• Applications of tissue culture technique in plant improvement and sustainable agriculture

Biotechnology: Definition and Scope

Biotechnology is the use of living organisms, cells or their parts (enzymes, genes) to make useful products and services for human welfare. The term was first used by Karl Ereky (1919). The European Federation of Biotechnology defines it as "the integration of natural sciences and engineering sciences in order to achieve the application of organisms, cells, parts thereof and molecular analogues for products and services".

Scope: (1) Agriculture — tissue culture plants, transgenic crops (Bt cotton), biofertilisers, biopesticides; (2) Medicine — insulin, vaccines, antibiotics, gene therapy, diagnostics; (3) Industry — enzymes, alcohol, organic acids; (4) Environment — bioremediation, sewage treatment, biogas; (5) Food — fermented foods, single-cell protein; (6) Forensics — DNA fingerprinting.

Plant Tissue Culture: Basic Terms

Plant tissue culture is growing plant cells, tissues or organs on a sterile nutrient medium under controlled conditions (in vitro). Gottlieb Haberlandt (1902) is the father of plant tissue culture.

1. Cellular totipotency: the ability of a single living plant cell to divide and regenerate into a whole plant. It is the basis of tissue culture.
2. Explant: the small piece of plant (leaf, shoot tip, node, root, anther, embryo) taken to start a culture.
3. Aseptic culture: culture kept free from all microbes (bacteria, fungi), done in sterile conditions.
4. Micropropagation: rapid multiplication of a large number of genetically identical plants (clones) from small explants in vitro, e.g. banana, sugarcane, orchids.
5. De-differentiation: mature, specialised cells lose their special features and return to a dividing, meristematic state, forming callus.
6. Re-differentiation: callus cells again become specialised to form organs or whole plants.
7. Morphogenesis: development of form — organs arise from callus either as shoots/roots (organogenesis) or as embryo-like structures (somatic embryogenesis).

Plant Tissue Culture Laboratory

A tissue culture laboratory has separate areas: washing area, media preparation room, sterilisation area, transfer (inoculation) room with laminar air flow, and a culture (growth) room with racks, lights (about 16 h light / 8 h dark) and temperature about 25 ± 2 °C.

1. Culture cabinets / growth chambers: keep cultures at controlled temperature, light and humidity.
2. Preparation room: for weighing chemicals, preparing stock solutions and media; has a balance, hot plate/stirrer, refrigerator.
3. Oven (hot-air oven): dry-heat sterilisation of glassware and metal instruments.
4. Autoclave: moist-heat sterilisation of media and glassware by steam under pressure.
5. Laminar air flow (LAF) cabinet: blows filtered, sterile air through a HEPA filter across the work area; inoculation is done here. UV lamp sterilises the surface before use.
6. pH meter: to adjust medium pH (usually 5.6–5.8) before adding agar and autoclaving.
7. Glassware: borosilicate (Borosil/Pyrex) flasks, test tubes, Petri dishes, culture bottles, pipettes, measuring cylinders.

Media: Basal Medium and MS Medium

A basal medium contains the basic nutrients for plant cell growth: inorganic salts (macro- and micronutrients), an iron source, vitamins, a carbon source (sucrose), and agar for solid medium. Growth regulators (auxins, cytokinins) are added according to need.

Murashige and Skoog (MS) medium, 1962 — developed by T. Murashige and F. Skoog for tobacco callus; the most widely used medium. It is rich in nitrate and ammonium.

Preparation: prepare concentrated stock solutions of macronutrients, micronutrients, iron and vitamins → mix required volumes → add sucrose and growth regulators → make up volume with distilled water → adjust pH to 5.8 → add agar and melt → dispense into culture vessels → autoclave at 121 °C for 15–20 min.

Methods of Sterilization

Physical methods:

1. Dry heat: hot-air oven at 160–180 °C for 2–3 hours; for glassware and metal instruments. Forceps and scalpels are also flamed after dipping in alcohol.
2. Moist heat (autoclave): steam at 121 °C and 15 lb/inch² (about 1.05 kg/cm²) pressure for 15–20 minutes; kills spores; for media, water and glassware.
3. Filter sterilization: heat-sensitive solutions (some vitamins, hormones, antibiotics) are passed through membrane filters of pore size 0.22 µm that hold back microbes.
4. UV radiation: UV lamps (about 260 nm) in the LAF and transfer room kill microbes on surfaces and in air.

Chemical methods (mainly for surface sterilization of explants and the working area):

1. Ethyl alcohol (70%): explant dipped for 30–60 seconds; also to wipe hands and the LAF.
2. Calcium hypochlorite and sodium hypochlorite (dilute bleach, about 1–2% NaOCl): explant treated for 10–20 minutes, then washed with sterile water.
3. Mercuric chloride (0.1% HgCl₂): strong sterilant for 2–10 minutes; highly toxic, must be washed well.
4. Hydrogen peroxide (H₂O₂, about 10%): mild sterilant.
5. Antibiotics (e.g. streptomycin, carbenicillin) and fungicides: added to control hidden contamination.

Types and Methods of In Vitro Culture

1. Embryo culture (whole plant culture): zygotic embryos are removed from seeds and grown on medium. Importance: rescues hybrid embryos that would abort (embryo rescue in wide crosses), overcomes seed dormancy, shortens the breeding cycle, and helps seedless fruit varieties.
2. Anther culture: anthers with immature pollen are cultured to produce haploid plants (androgenesis), first by Guha and Maheshwari (1964) in Datura. Importance: doubling haploids with colchicine gives homozygous pure lines quickly; useful in breeding and mutation studies.
3. Shoot tip (meristem) culture: tiny apical meristem (0.1–0.5 mm) is cultured. Importance: produces virus-free plants (e.g. potato, banana, sugarcane) and is used for micropropagation.
4. Callus culture: an unorganised mass of dividing cells (callus) is grown from explants on medium with auxin (2,4-D). Importance: source of somaclonal variation, cell suspension cultures for secondary metabolites, regeneration of plants and genetic transformation.
5. Protoplast culture: protoplasts (cells without walls) are isolated by enzymes (cellulase + pectinase) and cultured; they regenerate walls and divide. Importance: somatic hybridisation (protoplast fusion, e.g. pomato), direct gene transfer, study of membranes.
6. Synthetic (artificial) seeds: somatic embryos or shoot buds encapsulated in a gel such as sodium alginate (hardened in calcium chloride) to form beads that act like seeds. Advantages: easy storage and transport, direct sowing, clonal (uniform) plants, cheaper for rare/elite plants. Disadvantages: poor germination and conversion, embryos may dry or get contaminated, short storage life, costly technology.

Applications of Tissue Culture in Plant Improvement and Sustainable Agriculture

• Mass production of elite, disease-free planting material (banana, sugarcane, potato, teak, bamboo, orchids).
• Virus elimination by meristem culture.
• Haploid and pure-line production for faster breeding.
• Somaclonal variation and in vitro selection for disease or salt tolerance.
• Embryo rescue for wide hybrids; somatic hybrids.
• Germplasm conservation (cryopreservation at −196 °C in liquid nitrogen) and conservation of rare and endangered plants.
• Production of secondary metabolites (e.g. shikonin) without cutting wild plants.
• Base for genetic transformation and transgenic plants.

Likely exam questions

1. (Long, 8 marks) Describe the composition and preparation of MS medium. Add a note on the physical and chemical methods of sterilization used in plant tissue culture.
2. (Long, 8 marks) Explain anther culture, shoot tip culture and protoplast culture with their importance.
3. (Short, 4 marks) Define cellular totipotency and explant.
4. (Short, 4 marks) What are synthetic seeds? Give their advantages and disadvantages.
5. (Short, 4 marks) Write the role of laminar air flow and autoclave in a tissue culture laboratory.

Syllabus topics (RTMNU)

• Genetic Engineering/Gene Manipulation: Definition, scope and importance in plant biotechnology
• Tools - Enzymes: Restriction Endonucleases and types, DNA Polymerases and types, Ligases
• Vectors: Definition, characteristics and types; Plasmid as a cloning vector and expression vector; Shuttle vector
• Passenger DNA: DNA insert or Foreign DNA
• Methods for obtaining Passenger DNA: Splicing of parent DNA, Chemically synthesized DNA (sDNA), DNA Libraries: cDNA library and Genomic library
• Transgenic plants: Agrobacterium mediated gene Transfer: Structure of Ti-plasmid and mechanism of gene transfer
• Direct gene transfer mechanism: Electroporation, Particle bombardment gun method
• Examples: Bt-cotton and Golden rice
• Advantages and disadvantages of transgenic crop plants

Genetic Engineering: Definition, Scope and Importance

Genetic engineering (gene manipulation / recombinant DNA technology) is the deliberate alteration of an organism's genetic material by isolating a desired gene, joining it to a vector, and transferring it into another organism where it is expressed. The first recombinant DNA was made by Stanley Cohen and Herbert Boyer (1973).

Basic steps: (1) isolate the desired gene (passenger DNA); (2) cut it and the vector with the same restriction enzyme; (3) join them with ligase → recombinant DNA; (4) transfer into a host cell; (5) select the transformed cells; (6) multiply and express the gene.

Scope and importance in plant biotechnology: insect-resistant crops (Bt cotton), herbicide-tolerant crops, virus-resistant crops (papaya), improved nutrition (Golden rice), delayed ripening (Flavr Savr tomato), tolerance to drought and salinity, molecular farming (vaccines and medicines in plants), and basic study of gene function.

Tools: Enzymes

i. Restriction endonucleases ("molecular scissors") cut DNA at specific recognition sequences. They occur in bacteria as a defence against phages (restriction–modification system). Discovered by Arber, Smith and Nathans (Nobel 1978). Names come from the bacterium: EcoRI = Escherichia coli strain RY13, first enzyme.

Most type II sites are palindromes — read the same 5′→3′ on both strands. They make sticky (cohesive) ends with short single-stranded overhangs, e.g. EcoRI G↓AATTC, BamHI G↓GATCC, HindIII A↓AGCTT; or blunt ends, e.g. SmaI CCC↓GGG.

ii. DNA polymerases synthesise new DNA strands in the 5′→3′ direction using a template and a primer.

• Prokaryotic: DNA Pol I (Kornberg, 1956; removes RNA primers and fills gaps; its large part is the Klenow fragment), Pol II (DNA repair), Pol III (main replicating enzyme).
• Eukaryotic: Pol α (primase activity, starts replication), Pol δ and Pol ε (main replication of lagging and leading strands), Pol β (repair), Pol γ (mitochondrial DNA).
• Special: Taq polymerase from Thermus aquaticus — heat-stable, used in PCR; reverse transcriptase — makes DNA from RNA (used for cDNA).

iii. Ligases ("molecular glue") join DNA fragments by forming a phosphodiester bond between the 3′-OH of one nucleotide and the 5′-phosphate of the next. T4 DNA ligase (uses ATP) joins both sticky and blunt ends; E. coli ligase uses NAD⁺.

Tools: Vectors

A vector (cloning vehicle) is a DNA molecule that carries foreign DNA into a host cell and replicates there.

Characteristics of a good vector: (1) an origin of replication (ori); (2) selectable marker genes (e.g. ampicillin or tetracycline resistance); (3) unique restriction sites / multiple cloning site; (4) small size and easy isolation; (5) high copy number; (6) easy transfer into host.

Types: plasmids (pBR322, pUC19), bacteriophage λ vectors, cosmids, BAC (bacterial artificial chromosome), YAC (yeast artificial chromosome), Ti plasmid (plants), viral vectors.

Plasmid as a cloning vector: plasmids are small, circular, double-stranded, extra-chromosomal DNA of bacteria that replicate on their own. pBR322 (Bolivar and Rodriguez; 4361 bp) has ori, amp^R and tet^R genes and sites like BamHI inside tet^R. When foreign DNA is inserted into tet^R, the gene is inactivated (insertional inactivation): recombinant cells grow on ampicillin but not on tetracycline. pUC vectors use lacZ for blue–white screening.

Plasmid as an expression vector: designed not just to copy the gene but to make its protein. It carries a strong promoter, a ribosome binding site (Shine–Dalgarno sequence), the cloning site in the right reading frame and a terminator, e.g. pET vectors used to produce human insulin in E. coli.

ii. Shuttle vector: a vector that can replicate in two different hosts (e.g. E. coli and yeast, or E. coli and Agrobacterium/plant cells). It has two origins of replication and selectable markers for both hosts. DNA is cloned in E. coli (easy) and then "shuttled" to the other host, e.g. yeast episomal plasmids (YEp), binary Ti vectors.

Passenger DNA and Methods of Obtaining It

Passenger DNA (insert / foreign / target DNA) is the gene or DNA fragment to be cloned. Methods:

1. Splicing of parent DNA: total DNA of the source organism is cut with restriction enzymes; the fragment with the desired gene is separated (gel electrophoresis) and joined to a vector.
2. Chemically synthesised DNA (sDNA): when the protein or gene sequence is known, the gene is made in the laboratory by joining nucleotides one by one (phosphoramidite method in "gene machines"). H.G. Khorana (1970) first synthesised a gene (yeast alanine tRNA gene). Used for small genes like insulin chains.
3. DNA libraries: a collection of cloned DNA fragments representing the organism's DNA. (a) Genomic library — the whole genome is cut into fragments and each is cloned in vectors; contains introns, non-coding and regulatory DNA. (b) cDNA library — mRNA from a tissue is copied into complementary DNA (cDNA) by reverse transcriptase, made double-stranded and cloned; contains only expressed genes, without introns, so it can be expressed in bacteria.

Transgenic Plants: Agrobacterium-Mediated Gene Transfer

Agrobacterium tumefaciens is a soil bacterium that causes crown gall disease in dicots. It is called "nature's genetic engineer" because it naturally transfers part of its DNA into plant cells.

Structure of Ti (tumour-inducing) plasmid (about 200 kb):

1. T-DNA (transfer DNA, about 20 kb) bounded by 25 bp left and right border repeats; carries onc genes for auxin and cytokinin synthesis (cause tumour) and opine synthesis genes (opines are food for the bacterium).
2. Virulence (vir) region: genes virA, virB, virC, virD, virE, virG that cut and transfer T-DNA.
3. Opine catabolism region and an origin of replication.

Mechanism of gene transfer: (1) wounded plant cells release phenolics such as acetosyringone; (2) VirA senses it and activates VirG; (3) VirG switches on other vir genes; (4) VirD1/VirD2 nick the border sequences and a single-stranded T-strand is released with VirD2 attached; (5) VirE2 coats it; (6) it passes through a channel/pilus made by VirB proteins into the plant cell; (7) it enters the nucleus and integrates into plant chromosomes. For gene transfer, scientists use disarmed Ti plasmids (onc genes removed, desired gene + marker inserted between the borders), usually as a binary vector system.

Direct Gene Transfer: Electroporation and Particle Bombardment

Electroporation: protoplasts (or cells) mixed with DNA are given short high-voltage electric pulses; temporary pores form in the membrane and DNA enters. Useful for monocots like rice and maize.

Particle bombardment (gene gun / biolistics): developed by J. Sanford (1987). Tiny gold or tungsten particles (about 1 µm) are coated with DNA and shot at high speed (helium pressure) into cells, tissues or embryos. DNA enters the nucleus and integrates. Works for any plant, including cereals; also for chloroplast transformation.

Examples: Bt Cotton and Golden Rice

Bt cotton: carries cry genes (e.g. cry1Ac; Bollgard II has cry1Ac + cry2Ab) from the soil bacterium Bacillus thuringiensis. The gene makes an inactive crystal protein (protoxin). When a bollworm larva eats the leaf, the alkaline gut activates it into a toxin that binds to gut cells, makes pores, and kills the larva. It protects against American bollworm. Approved in India in 2002 by GEAC.

Golden rice: developed by Ingo Potrykus and Peter Beyer (2000) to fight vitamin A deficiency. Two genes — psy (phytoene synthase, from daffodil; from maize in Golden Rice 2) and crtI (phytoene desaturase from the bacterium Erwinia/Pantoea) — complete the β-carotene pathway in the endosperm, giving yellow-golden grains. β-carotene is converted into vitamin A in the body.

Advantages and Disadvantages of Transgenic Crops

Likely exam questions

1. (Long, 8 marks) What are restriction endonucleases? Describe their types. Add a note on DNA ligase and DNA polymerases.
2. (Long, 8 marks) Describe the structure of the Ti plasmid and the mechanism of Agrobacterium-mediated gene transfer.
3. (Short, 4 marks) What is a shuttle vector?
4. (Short, 4 marks) Distinguish between a cDNA library and a genomic library.
5. (Short, 4 marks) Write a short note on Golden rice.

Syllabus topics (RTMNU)

• Genome: Definition, organization of genome, Types: Prokaryotic and Eukaryotic
• DNA: Watson and Crick's model of DNA
• Replication of DNA: Semi-conservative method in prokaryotes
• RNA: Types of RNAs, Clover leaf model of t-RNA
• Concept of Gene: Classical (Cistron, Muton and Recon)
• Modern: One gene one enzyme, one gene one polypeptide and gene as a transcriptional unit
• Organization of Genes: Structure of eukaryotic gene; unique and repetitive DNA

Genome: Definition, Organization and Types

Genome is the complete set of genetic material (DNA, or RNA in some viruses) of an organism, i.e. one full set of its genes and non-coding sequences. The term was given by H. Winkler (1920).

Organisation of eukaryotic chromatin: DNA (2 nm) → nucleosome — about 146 bp of DNA wound 1.65 turns around a histone octamer (two each of H2A, H2B, H3, H4), with H1 on the linker DNA ("beads on a string", 11 nm) → 30 nm solenoid fibre → looped domains on a protein scaffold → condensed chromosome at metaphase. Loosely packed, active chromatin is euchromatin; densely packed, inactive chromatin is heterochromatin.

DNA: Watson and Crick Model

In 1953 J.D. Watson and F.H.C. Crick proposed the double helix model of DNA, using X-ray diffraction data of Rosalind Franklin and Maurice Wilkins and Chargaff's base rules (Nobel 1962).

1. DNA has two polynucleotide chains coiled around a common axis into a right-handed double helix.
2. The chains are antiparallel — one runs 5′→3′, the other 3′→5′.
3. The sugar–phosphate backbone (deoxyribose + phosphate, joined by 3′–5′ phosphodiester bonds) is outside; nitrogen bases are inside.
4. Bases pair by hydrogen bonds: A = T (two H-bonds) and G ≡ C (three H-bonds) — complementary base pairing. So A = T and G = C in amount (Chargaff's rule).
5. Diameter of the helix is 2 nm (20 Å); one complete turn is 3.4 nm (34 Å) and has 10 base pairs; distance between adjacent base pairs is 0.34 nm.
6. The helix has a major groove and a minor groove. This form is called B-DNA.

Replication of DNA: Semi-Conservative Method in Prokaryotes

Semi-conservative replication: each daughter DNA has one old (parental) strand and one newly made strand. Proved by Meselson and Stahl (1958): E. coli grown in heavy nitrogen (¹⁵N) was moved to ¹⁴N; after one generation DNA had intermediate (hybrid) density in CsCl density gradient centrifugation; after two generations, half hybrid and half light.

Steps in E. coli:

1. Initiation: replication begins at a single origin (oriC). Initiator proteins (DnaA) open the DNA.
2. Unwinding: helicase breaks H-bonds and separates strands, forming a Y-shaped replication fork; single-strand binding proteins (SSB) keep strands apart; DNA gyrase (topoisomerase) removes the supercoiling ahead.
3. Primer synthesis: primase makes a short RNA primer, because DNA polymerase cannot start a chain on its own.
4. Elongation: DNA polymerase III adds nucleotides in the 5′→3′ direction. The leading strand is made continuously towards the fork; the lagging strand is made discontinuously as short Okazaki fragments (1000–2000 nucleotides) away from the fork.
5. Primer removal and joining: DNA polymerase I removes RNA primers and fills gaps with DNA; DNA ligase seals the nicks.
6. Termination: the two forks move in opposite directions (bidirectional, "theta" shape) and meet at the ter region.

RNA: Types of RNA and Clover Leaf Model of tRNA

RNA is usually single-stranded, has ribose sugar and uracil (U) in place of thymine.

1. mRNA (messenger RNA): carries the genetic message (codons) from DNA to ribosomes; about 3–5% of cell RNA.
2. tRNA (transfer RNA / soluble RNA): carries specific amino acids to the ribosome and reads codons with its anticodon; about 10–15%.
3. rRNA (ribosomal RNA): structural and catalytic part of ribosomes (e.g. 23S rRNA acts as peptidyl transferase); about 80%.
4. Others: snRNA (splicing), miRNA and siRNA (gene silencing).

Clover leaf model was proposed by R.W. Holley (1965) for yeast alanine tRNA. tRNA has about 75–90 nucleotides folded by internal base pairing into a two-dimensional clover leaf with four arms:

1. Acceptor (amino acid) arm: ends in CCA-3′; the amino acid attaches to the terminal adenosine.
2. DHU (D) arm: loop with dihydrouridine; recognition site for aminoacyl-tRNA synthetase.
3. Anticodon arm: loop with three bases (anticodon) that pair with the codon on mRNA.
4. Variable arm: varies in length between tRNAs.
5. TΨC arm: has thymine, pseudouridine and cytosine; binds to the ribosome.

In three dimensions tRNA is folded into an L-shape. It contains many unusual (modified) bases.

Concept of Gene: Classical and Modern

Classical concept: Mendel's "factors" were later called genes (Johannsen, 1909). Genes were thought to be bead-like units of function, mutation and recombination. Seymour Benzer (1955–61), working on the rII region of T4 phage, showed the gene can be subdivided and gave three terms:

1. Cistron: the unit of function — the segment of DNA coding for one polypeptide; identified by the cis–trans complementation test.
2. Muton: the smallest unit of DNA that can mutate — as small as one nucleotide pair.
3. Recon: the smallest unit of recombination — two adjacent nucleotide pairs can recombine.

Modern concept:

1. One gene – one enzyme (Beadle and Tatum, 1941, on the mould Neurospora crassa): each gene controls the synthesis of one enzyme.
2. One gene – one polypeptide: since many proteins (e.g. haemoglobin) have more than one type of polypeptide chain, each coded by a separate gene, the idea was refined to one gene → one polypeptide.
3. Gene as a transcriptional unit: today a gene is defined as a segment of DNA that is transcribed into one functional RNA (mRNA, tRNA or rRNA), together with its regulatory sequences (promoter, terminator). This includes genes that make RNA but no protein, and genes with introns.

Organization of Genes: Eukaryotic Gene Structure; Unique and Repetitive DNA

Structure of a eukaryotic gene (5′ → 3′):

1. Promoter: upstream site where RNA polymerase II binds; contains the TATA box (about −25 to −30), CAAT box (about −75) and GC box.
2. Enhancers / silencers: regulatory sequences, often far away, that increase or decrease transcription.
3. Transcription start site (+1) and 5′ untranslated region (UTR).
4. Exons (coding sequences) interrupted by introns (non-coding; removed by splicing). The first exon carries the start codon ATG.
5. 3′ UTR with the polyadenylation signal (AATAAA), then the terminator region.

Unique (single-copy) DNA: sequences present only once in the haploid genome; most protein-coding genes belong here.

Repetitive DNA: sequences present in many copies, shown by DNA re-association (C₀t) studies of Britten and Kohne (1968).

• Highly repetitive DNA: short sequences repeated millions of times in tandem, e.g. satellite DNA at centromeres and telomeres; minisatellites and microsatellites (used in DNA fingerprinting).
• Moderately repetitive DNA: repeated tens to thousands of times — rRNA genes, tRNA genes, histone genes, and dispersed transposable elements (SINEs such as Alu, LINEs).

Likely exam questions

1. (Long, 8 marks) Describe the Watson and Crick model of DNA. Explain the semi-conservative replication of DNA in prokaryotes.
2. (Long, 8 marks) Explain the classical and modern concepts of the gene.
3. (Short, 4 marks) Describe the clover leaf model of tRNA.
4. (Short, 4 marks) Distinguish between prokaryotic and eukaryotic genomes.
5. (Short, 4 marks) What is repetitive DNA? Name its types.

Syllabus topics (RTMNU)

• Types of Genes: Overlapping gene, split gene, jumping gene, Cryptic gene, Pseudogenes and Gene families in eukaryotes
• Genetic Code: Definition and characteristics of genetic code
• Gene Expression in Prokaryotes: Central dogma of protein synthesis, Transcription and Translation
• Regulation of Gene Action in Prokaryotes: Lac Operon Model
• Genomics and its applications in production of Transgenic plants
• Proteomics and its applications

Types of Genes

1. Overlapping genes: two or more genes that share the same DNA sequence, read in different reading frames or partly overlapping. First found by F. Sanger (1977) in the phage φX174: gene E lies inside gene D; gene B inside gene A. They save space in small genomes of viruses.
2. Split genes (interrupted genes): genes whose coding sequences (exons) are separated by non-coding sequences (introns). Discovered by P. Sharp and R. Roberts (1977) in adenovirus (Nobel 1993). Introns are removed from pre-mRNA by splicing. Common in eukaryotes; e.g. the ovalbumin gene has 7 introns. Alternative splicing allows one gene to make several proteins.
3. Jumping genes (transposons / transposable elements): DNA segments that can move from one position to another in the genome. Discovered by Barbara McClintock in maize (Ac–Ds system controlling kernel colour), Nobel 1983. In bacteria — insertion sequences (IS) and transposons (Tn). They cause mutations, chromosome breaks and genome evolution, and are used as tools for gene tagging.
4. Cryptic genes: silent genes that are present but normally not expressed; they can be activated by mutation or recombination, e.g. the bgl operon of E. coli. They are a reserve of genetic variation.
5. Pseudogenes: non-functional copies of normal genes that have lost the ability to make a working product because of mutations (stop codons, frameshifts, missing promoter). Shown by the symbol ψ, e.g. ψβ1 in the β-globin cluster. Processed pseudogenes lack introns and arise when mRNA is reverse-transcribed and inserted into the genome.
6. Gene families: groups of genes with similar sequence and related function that arose by duplication of an ancestral gene, e.g. the globin family (α- and β-globin clusters), histone genes, rRNA genes, and immunoglobulin genes. Members may be clustered or dispersed and may include pseudogenes.

Genetic Code: Definition and Characteristics

Genetic code is the set of rules by which the sequence of nucleotides in mRNA is translated into the sequence of amino acids in a protein. Each amino acid is coded by a group of three bases called a codon. The first codon (UUU = phenylalanine) was decoded by Nirenberg and Matthaei (1961); Khorana and Holley completed the work (Nobel 1968). The term "triplet code" was supported by Crick (1961).

1. Triplet: three bases form one codon; 4³ = 64 codons.
2. Start codon: AUG (methionine; formyl-methionine in prokaryotes) begins translation.
3. Stop (nonsense) codons: UAA, UAG, UGA do not code any amino acid; 61 codons code for 20 amino acids.
4. Degenerate: most amino acids have more than one codon (e.g. leucine has 6); only Met (AUG) and Trp (UGG) have one each.
5. Non-overlapping: each base belongs to only one codon.
6. Comma-less: no gaps or punctuation between codons; read continuously.
7. Unambiguous: one codon codes only one amino acid.
8. Universal: the same code is used in almost all organisms (exceptions: mitochondria, some protozoa — e.g. UGA codes tryptophan in mitochondria).
9. Polarity: read in the 5′→3′ direction.
10. Wobble: the third base of the codon pairs loosely with the anticodon (Crick's wobble hypothesis, 1966), so one tRNA can read more than one codon.

Gene Expression in Prokaryotes: Central Dogma, Transcription and Translation

Central dogma (Crick, 1958): genetic information flows DNA → RNA → Protein — DNA replicates, is transcribed into RNA, and RNA is translated into protein. Reverse transcription (RNA → DNA) in retroviruses was found later (Temin and Baltimore, 1970). In prokaryotes transcription and translation are coupled (happen together in the cytoplasm) and mRNA is often polycistronic.

Transcription — synthesis of RNA from a DNA template by RNA polymerase. In E. coli one RNA polymerase (core enzyme α₂ββ′ω + sigma (σ) factor = holoenzyme) makes all RNAs.

1. Initiation: σ factor recognises the promoter — the −35 sequence (TTGACA) and the −10 Pribnow box (TATAAT). The enzyme binds and opens the DNA.
2. Elongation: σ leaves; the core enzyme moves along the template strand (3′→5′) and makes RNA in the 5′→3′ direction using ribonucleoside triphosphates.
3. Termination: (a) rho-independent — a GC-rich hairpin followed by a string of U's makes the enzyme fall off; (b) rho-dependent — the rho protein (a helicase) releases the RNA.

Translation — synthesis of a polypeptide on the 70S ribosome (30S + 50S) using the mRNA code.

1. Activation of amino acids: amino acid + ATP + tRNA → aminoacyl-tRNA, catalysed by aminoacyl-tRNA synthetase.
2. Initiation: the 30S subunit binds mRNA at the Shine–Dalgarno sequence; initiator fMet-tRNA pairs with AUG at the P site; initiation factors (IF1, IF2, IF3) and GTP help; then the 50S subunit joins.
3. Elongation: the next aminoacyl-tRNA enters the A site (with EF-Tu and GTP); peptidyl transferase (23S rRNA) forms a peptide bond; translocation by EF-G moves the ribosome one codon along (tRNA shifts from A to P, then to E site and exits).
4. Termination: when a stop codon reaches the A site, release factors (RF1, RF2, RF3) cause release of the polypeptide; the ribosome splits into subunits.

Regulation of Gene Action in Prokaryotes: Lac Operon Model

An operon is a group of structural genes controlled together by one promoter and operator. The lac operon of E. coli was proposed by F. Jacob and J. Monod (1961) (Nobel 1965). It controls lactose use and is an inducible operon under negative control.

Components:

1. Regulator gene (lacI): makes the repressor protein (always made).
2. Promoter (P): site where RNA polymerase binds.
3. Operator (O): switch region where the repressor binds.
4. Structural genes: lacZ (β-galactosidase — splits lactose into glucose and galactose), lacY (permease — brings lactose into the cell), lacA (transacetylase).

Working:

• Lactose absent (operon OFF): the active repressor binds the operator and blocks RNA polymerase; structural genes are not transcribed.
• Lactose present (operon ON): lactose is changed to allolactose, the inducer. It binds the repressor, changes its shape, and the repressor leaves the operator. RNA polymerase transcribes lacZ, lacY, lacA as one polycistronic mRNA; enzymes are made. When lactose is used up, the repressor becomes active again and switches the operon off.
• Positive control: when glucose is low, cAMP rises and the CAP–cAMP complex binds near the promoter and boosts transcription; when glucose is present, the operon stays low (catabolite repression).

Genomics and Its Applications in Transgenic Plants; Proteomics and Its Applications

Genomics is the study of the complete genome of an organism — its sequencing, mapping, gene content and function. The term was coined by T. Roderick (1986). Branches: structural genomics (sequencing and mapping), functional genomics (gene function and expression), comparative genomics (comparing genomes). Plant genomes sequenced include Arabidopsis thaliana (2000) and rice.

Applications in producing transgenic plants:

1. Identifying useful genes (pest resistance, drought and salt tolerance, nutrition) for transfer.
2. Finding suitable promoters (tissue-specific, inducible) to express the transgene where needed.
3. Molecular markers and marker-assisted selection to track the transgene in breeding.
4. Checking the site and copy number of transgene insertion and confirming no harmful changes.
5. Genome editing tools (e.g. CRISPR–Cas9) to change specific genes precisely.

Proteomics is the large-scale study of the proteome — the complete set of proteins expressed by a cell, tissue or organism at a given time (term proteome by Marc Wilkins, 1994). Techniques: two-dimensional gel electrophoresis, mass spectrometry (e.g. MALDI-TOF), protein microarrays, bioinformatics.

Applications: finding the function of proteins; identifying proteins made under stress (drought, pathogens) for crop improvement; checking the expression and safety (allergenicity) of proteins in transgenic plants; disease biomarkers and drug targets in medicine; studying protein–protein interactions.

Likely exam questions

1. (Long, 8 marks) Describe the process of transcription and translation in prokaryotes.
2. (Long, 8 marks) Explain the lac operon model of gene regulation in E. coli with a labelled diagram.
3. (Short, 4 marks) Write any four characteristics of the genetic code.
4. (Short, 4 marks) What are split genes and jumping genes?
5. (Short, 4 marks) What is proteomics? Give its applications.