Molecular Biology & Bioinformatics BBO6T16

Syllabus topics (RTMNU)

• Molecular Biology: History and scope of Molecular Biology; DNA as Genetic Material; Watson and Crick's model of DNA
• Genome size and complexity: Prokaryotic (bacterial chromosomes, plasmids) and eukaryotic (chromosomes and organellar genomes, mobile elements, repetitive sequences, eu- and hetero-chromatin and gene families)
• DNA replication in bacteria and eukaryotes: Mechanism of replication, proteins and enzymes involved in replication
• Chromosome organization: Packaging of DNA in chromosome

History and scope of molecular biology

Molecular biology studies life at the level of molecules — DNA, RNA and proteins — and how genetic information is stored, copied and expressed. The term was used by Warren Weaver (1938). Milestones: Miescher discovered "nuclein" (1869); Griffith's transformation (1928); Avery, MacLeod and McCarty (1944) showed DNA is the transforming principle; Hershey and Chase (1952) confirmed DNA is the genetic material of phage; Chargaff's rules (A = T, G = C); Watson and Crick's double helix (1953); Meselson and Stahl — semi-conservative replication (1958); Crick's central dogma (1958); Jacob and Monod's lac operon (1961); genetic code cracked by Nirenberg, Khorana and others (1961–66); restriction enzymes and recombinant DNA (1970s); Sanger sequencing (1977); PCR (1983); Arabidopsis genome (2000), human genome (2003).

Scope: genetic engineering and transgenic crops, molecular markers in plant breeding, diagnostics, gene therapy, forensic DNA profiling, evolution and taxonomy (DNA barcoding), genomics and bioinformatics.

DNA as the genetic material

• Griffith (1928): heat-killed virulent S-type Streptococcus pneumoniae + live non-virulent R-type injected into mice → mice died, live S-type recovered — "transforming principle".
• Avery, MacLeod, McCarty (1944): purified extract of S cells; transformation destroyed by DNase but not by protease or RNase → DNA is the transforming principle.
• Hershey and Chase (1952): T2 phage labelled with ³²P (DNA) or ³⁵S (protein); after infection and blending, ³²P entered bacteria and appeared in progeny phage; ³⁵S stayed outside → DNA is the genetic material. (In some plant viruses such as TMV, RNA is the genetic material — Fraenkel-Conrat.)

Watson and Crick model of DNA (B-DNA)

• Two polynucleotide strands wound into a right-handed double helix around a common axis; strands are antiparallel (5′→3′ and 3′→5′).
• Sugar–phosphate backbone outside; nitrogen bases inside, stacked; bases paired by hydrogen bonds — A=T (2 H-bonds), G≡C (3 H-bonds) — complementary base pairing (purine with pyrimidine), so the diameter is uniform, about 2 nm (20 Å).
• One turn = 3.4 nm with about 10 base pairs (0.34 nm between pairs; 10.5 bp per turn in solution).
• Major and minor grooves, where proteins bind.
• Based on Franklin and Wilkins' X-ray diffraction data and Chargaff's rules. Nobel Prize 1962 (Watson, Crick, Wilkins).

Genome size and complexity

Prokaryotic genome: usually a single circular, double-stranded chromosome (E. coli about 4.6 Mb, about 4,300 genes) in the nucleoid, supercoiled with histone-like proteins (HU, H-NS); genes closely packed, few repeats, no introns; often organised in operons. Plasmids: small, circular, self-replicating extrachromosomal DNA carrying non-essential genes (antibiotic resistance, F factor, Ti plasmid of Agrobacterium); used as cloning vectors.

Eukaryotic genome: several linear chromosomes in the nucleus plus organellar genomes; much larger and more complex (Arabidopsis about 135 Mb, rice about 430 Mb, maize about 2.3 Gb, wheat about 16 Gb). Features:

• Organellar genomes: chloroplast DNA (cpDNA, circular, about 120–160 kb, codes for rRNAs, tRNAs, photosynthesis genes such as rbcL) and mitochondrial DNA (plants: large, 200–2,400 kb, recombining); maternally inherited in most plants.
• Repetitive sequences: highly repetitive (satellite DNA, telomeric and centromeric repeats), moderately repetitive (rRNA genes, transposons) and unique (single-copy) sequences. Studied by C₀t (reassociation) curves.
• Mobile elements (transposons): "jumping genes" discovered by Barbara McClintock in maize (Ac/Ds); DNA transposons (cut-and-paste) and retrotransposons (copy via RNA) — make up most of large plant genomes (about 85% of maize).
• Euchromatin and heterochromatin: euchromatin is lightly stained, less condensed, gene-rich and active; heterochromatin is darkly stained, highly condensed, gene-poor and inactive — constitutive (centromeres, telomeres) or facultative (e.g. inactive X).
• Gene families: groups of related genes from duplication (e.g. globin genes, histone genes, rRNA genes, chlorophyll a/b binding protein genes); may include pseudogenes.
• C-value paradox: genome size (C-value) does not match organism complexity, mainly due to non-coding repetitive DNA.

DNA replication in bacteria and eukaryotes

Replication is semi-conservative (Meselson and Stahl, 1958, ¹⁵N/¹⁴N and CsCl density gradient), bidirectional from an origin, and semi-discontinuous: the leading strand is made continuously 5′→3′; the lagging strand in short Okazaki fragments (1,000–2,000 nt in bacteria, 100–200 nt in eukaryotes).

Differences: bacteria have one origin and one replicon, fast (about 1,000 nt s^–1), throughout the cell cycle, termination at ter sites (Tus protein); eukaryotes have many origins (replicons), slower (about 50 nt s^–1), only in S phase, licensing so each origin fires once, nucleosomes must be disassembled and reassembled, and linear ends need telomerase.

Chromosome organisation — packaging of DNA

About 2 m of DNA in a human cell fits into a nucleus of about 6 µm by hierarchical folding:

1. Nucleosome ("beads on a string", 11 nm fibre): about 146–147 bp of DNA wrapped 1.65 turns around a histone octamer (2 each of H2A, H2B, H3, H4); linker DNA between beads; H1 binds at entry/exit (chromatosome). Packing about 6-fold.
2. 30 nm fibre (solenoid): nucleosomes coiled with about 6 per turn, stabilised by H1 (about 40-fold).
3. Looped domains: 30 nm fibre forms loops (about 40–100 kb) attached to a non-histone protein scaffold/nuclear matrix (300 nm).
4. Further coiling to 700 nm chromatid and the metaphase chromosome (1,400 nm) — overall about 10,000-fold compaction.

Likely exam questions

1. (Long, 8 marks) Describe the mechanism of DNA replication in prokaryotes with the enzymes involved. How does eukaryotic replication differ?
2. (Long, 8 marks) Explain the organisation of prokaryotic and eukaryotic genomes. Add a note on repetitive DNA and transposons.
3. (Short, 4 marks) Describe the Watson and Crick model of DNA.
4. (Short, 4 marks) Explain the nucleosome model of DNA packaging.
5. (Short, 4 marks) Distinguish between euchromatin and heterochromatin.

Syllabus topics (RTMNU)

• Transcription in bacteria and eukaryotes: Initiation, elongation and termination; differences between bacterial and eukaryotic transcription; Promoter
• Post-transcriptional modifications of mRNA in eukaryotes: RNA splicing and RNA editing
• Structure of tRNA and rRNA
• Introduction to small RNAs and long non-coding RNAs
• Regulation of transcription: (a) Bacterial: Operon model – induction and repression, transcription factor, sigma factor substitution; (b) Eukaryotic: Activation and repression of transcription, DNA methylation

Transcription in bacteria

Transcription is the synthesis of RNA from a DNA template (template/antisense strand read 3′→5′; RNA made 5′→3′; no primer needed). Bacteria have one RNA polymerase: core enzyme α₂ββ′ω + σ (sigma) factor = holoenzyme.

1. Initiation: σ factor recognises the promoter — consensus −35 box (TTGACA) and −10 box/Pribnow box (TATAAT); closed complex → open complex (about 13 bp unwound) → first nucleotides joined; after about 10 nt σ is released (promoter clearance).
2. Elongation: core enzyme moves along, transcription bubble about 17 bp, RNA–DNA hybrid about 8 bp; about 40–80 nt s^–1.
3. Termination: (a) Rho-independent (intrinsic) — a GC-rich inverted repeat forms a hairpin followed by a run of U's; (b) Rho-dependent — Rho protein (a helicase) binds the rut site on RNA, moves along and releases the RNA.

Transcription in eukaryotes and differences from bacteria

Eukaryotes have three nuclear RNA polymerases — Pol I (28S, 18S, 5.8S rRNA), Pol II (mRNA, most snRNAs, miRNAs), Pol III (tRNA, 5S rRNA, U6 snRNA); plants also have Pol IV and Pol V (small-RNA silencing). Pol II promoters: core promoter with TATA box (about −25 to −30), Inr, plus proximal elements (CAAT box, GC box) and distant enhancers/silencers. Initiation needs general transcription factors (TFIID with TATA-binding protein, TFIIA, B, E, F, H) forming the pre-initiation complex; TFIIH phosphorylates the CTD of Pol II to start elongation. Termination of Pol II is coupled to cleavage at the poly(A) signal (AAUAAA).

Post-transcriptional modifications of eukaryotic mRNA

• 5′ capping: 7-methylguanosine joined by a 5′–5′ triphosphate bond — protects from exonucleases, helps export and ribosome binding.
• 3′ polyadenylation: cleavage about 10–30 nt after AAUAAA and addition of 100–250 A's by poly(A) polymerase — stability and translation.
• RNA splicing: removal of introns and joining of exons by the spliceosome (snRNPs U1, U2, U4, U5, U6). Introns begin with GU and end with AG (GU–AG rule) and have a branch-point A; two transesterification reactions release the intron as a lariat. Alternative splicing makes several proteins from one gene. Self-splicing introns (group I, II) need no proteins (ribozymes).
• RNA editing: change of nucleotide sequence after transcription. In plant mitochondria and chloroplasts, C → U editing is common (hundreds of sites) and often creates start codons or changes amino acids; in mammals, apolipoprotein B mRNA C → U editing gives a shorter protein in intestine; A → I editing by ADAR enzymes.

Structure of tRNA and rRNA

tRNA: 73–93 nucleotides; clover-leaf secondary structure (Holley, 1965) with: acceptor stem ending in 3′-CCA (amino acid attached to A); D-arm (dihydrouridine); anticodon arm with the anticodon loop pairing with the mRNA codon; variable arm; TΨC arm (binds ribosome). Contains modified bases (Ψ, D, inosine). Tertiary structure is an inverted L-shape.

rRNA: the structural and catalytic core of ribosomes. Prokaryotes: 23S and 5S (large 50S subunit), 16S (small 30S subunit; its 3′ end pairs with the Shine–Dalgarno sequence). Eukaryotes: 28S (25S in plants), 5.8S and 5S (60S subunit), 18S (40S subunit). rRNAs are highly folded with stems and loops; 23S/28S rRNA acts as the peptidyl transferase ribozyme. 16S/18S rRNA sequences are used in phylogeny.

Small RNAs and long non-coding RNAs

• microRNAs (miRNAs, 20–24 nt): from hairpin precursors cut by DICER-LIKE 1 (in plants); loaded on ARGONAUTE (RISC) and silence target mRNAs by cleavage (in plants, near-perfect pairing) or translational repression; control development (e.g. miR165/166 in leaf polarity, miR172 in flowering).
• small interfering RNAs (siRNAs, 21–24 nt): from long double-stranded RNA; defence against viruses and transposons; 24-nt siRNAs direct DNA methylation (RNA-directed DNA methylation, RdDM) via Pol IV/Pol V.
• Long non-coding RNAs (lncRNAs, > 200 nt): not translated; regulate genes by guiding chromatin modifiers, acting as decoys/sponges or scaffolds — e.g. COOLAIR and COLDAIR in Arabidopsis regulate FLC during vernalisation; XIST in mammalian X-inactivation.

Regulation of transcription — bacterial

Operon model (Jacob and Monod, 1961; Nobel 1965): a cluster of structural genes transcribed together under one promoter and an operator, controlled by a regulator gene.

• Inducible system — lac operon (catabolic): genes lacZ (β-galactosidase), lacY (permease), lacA (transacetylase). Without lactose, the lacI repressor binds the operator and blocks transcription. Lactose (as allolactose, the inducer) binds the repressor and inactivates it → transcription ON. Positive control: when glucose is low, cAMP–CAP (CRP) binds near the promoter and increases transcription (catabolite repression when glucose is present).
• Repressible system — trp operon (anabolic): five genes for tryptophan synthesis. The repressor is inactive alone; when tryptophan (co-repressor) is abundant it binds the repressor, which then binds the operator → transcription OFF. Fine control by attenuation (leader peptide, terminator vs anti-terminator hairpin).
• Transcription factors in bacteria are activators (CAP, AraC) and repressors (LacI, TrpR) binding specific DNA sites.
• Sigma factor substitution: different σ factors recognise different promoter sets, switching groups of genes on — e.g. σ⁷⁰ (housekeeping), σ³² (heat shock), σ⁵⁴ (nitrogen), σ^S (stationary phase); in Bacillus subtilis sporulation a cascade of σ factors; phage SPO1 uses its own σ factors for early, middle and late genes.

Regulation of transcription — eukaryotic

• Activation: activator proteins (with DNA-binding domains such as zinc finger, helix-turn-helix, leucine zipper, MADS box, and activation domains) bind enhancers/promoter elements and recruit the Mediator complex, general factors and chromatin remodellers; histone acetyltransferases (HATs) acetylate histones, loosening chromatin (open, active). Plant examples: MYB, bHLH, WRKY, MADS-box (ABC model of flower) factors; hormone-responsive factors.
• Repression: repressors compete with activators, block activation domains or recruit co-repressors and histone deacetylases (HDACs) → compact, silent chromatin; Polycomb proteins (H3K27 methylation) silence genes such as FLC.
• DNA methylation: addition of a methyl group to cytosine (5-methylcytosine) by DNA methyltransferases. In plants it occurs in CG, CHG and CHH contexts (MET1, CMT3, DRM2). Methylated promoters bind methyl-binding proteins and attract HDACs → gene silencing; silences transposons; heritable epigenetic marks (e.g. epialleles, imprinting); removed by demethylases (ROS1, DEMETER).

Likely exam questions

1. (Long, 8 marks) Describe the process of transcription in prokaryotes. How does it differ from eukaryotic transcription?
2. (Long, 8 marks) Explain the lac operon as an inducible system and the trp operon as a repressible system.
3. (Short, 4 marks) Describe the clover-leaf structure of tRNA.
4. (Short, 4 marks) What is RNA splicing? Explain with the role of the spliceosome.
5. (Short, 4 marks) Write a note on DNA methylation and gene regulation.

Syllabus topics (RTMNU)

• Genetic code: Characteristics, codon usage and bias, Wobble hypothesis
• Protein structure: Overview of amino acids and peptide bond; primary, secondary, tertiary and quaternary structure of protein
• Ribosome: Structure and functions of 70S and 80S ribosomes
• Translation (bacterial and eukaryotic): Pre-initiation, initiation, elongation and termination; overview of translation in chloroplasts and mitochondria
• Protein modification: De-formylation, cleavage of terminal amino acids and protein splicing
• Protein folding: Mechanism, role of chaperones and assembly of proteins

Genetic code — characteristics, codon usage and bias

The genetic code is the set of rules relating mRNA base triplets (codons) to amino acids. 4³ = 64 codons: 61 sense codons for 20 amino acids and 3 stop codons (UAA ochre, UAG amber, UGA opal).

1. Triplet (Crick and Brenner frameshift experiments, 1961).
2. Non-overlapping and commaless — read continuously from a fixed start.
3. Degenerate — most amino acids have more than one codon (Leu, Ser, Arg six each; Met and Trp only one).
4. Unambiguous — one codon = one amino acid.
5. Start codon AUG (Met; fMet in bacteria); rarely GUG.
6. Nearly universal — exceptions in mitochondria (UGA = Trp; AUA = Met in animals) and some protozoa (UAA/UAG = Gln).
7. Colinear with the polypeptide; read 5′→3′.

Codon usage and bias: synonymous codons are not used equally; each organism prefers certain codons, which matches its most abundant tRNAs (e.g. highly expressed genes use preferred codons). Codon bias affects translation speed and accuracy; for expressing a foreign gene (e.g. Bt cry gene in cotton) the gene is codon-optimised for the host plant.

Wobble hypothesis (Crick, 1966)

The first two bases of the codon pair strictly with the anticodon, but the third base of the codon (3′) pairs loosely with the first base (5′) of the anticodon. Permitted wobble pairs: G–U, and inosine (I) in the anticodon pairs with U, C or A. So one tRNA can read several codons, and fewer than 61 tRNAs (minimum about 32) are needed.

Protein structure — amino acids and peptide bond; levels of structure

Amino acids have an α-carbon bearing –NH₂, –COOH, H and a side chain (R). 20 standard amino acids: non-polar (Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Met), polar uncharged (Ser, Thr, Cys, Tyr, Asn, Gln), acidic (Asp, Glu), basic (Lys, Arg, His). A peptide bond (–CO–NH–) forms between the carboxyl of one and the amino group of the next with loss of water; it is planar and rigid (partial double-bond character), trans.

• Primary: linear sequence of amino acids (Sanger sequenced insulin, 1953); determines all higher levels.
• Secondary: local folding by H-bonds between backbone C=O and N–H — α-helix (Pauling, right-handed, 3.6 residues per turn, 0.54 nm pitch) and β-pleated sheet (parallel/antiparallel); turns and loops.
• Tertiary: overall 3-D folding of one chain by hydrophobic interactions, H-bonds, ionic bonds and disulphide bridges; forms domains (e.g. myoglobin).
• Quaternary: association of two or more polypeptide subunits (e.g. haemoglobin α₂β₂; RuBisCO L₈S₈ — 8 large chloroplast-coded + 8 small nucleus-coded subunits).

Ribosome — structure and functions of 70S and 80S

Functional sites: mRNA-binding site on the small subunit; A site (aminoacyl-tRNA), P site (peptidyl-tRNA), E site (exit); peptidyl transferase centre in the large subunit (rRNA ribozyme); exit tunnel for the growing chain. Several ribosomes on one mRNA form a polysome.

Translation in bacteria and eukaryotes

1. Pre-initiation (charging of tRNA): aminoacyl-tRNA synthetases attach each amino acid to its tRNA using ATP: aa + ATP → aa-AMP; aa-AMP + tRNA → aa-tRNA + AMP. In bacteria the initiator Met-tRNA_f is formylated to fMet-tRNA_f.
2. Initiation: Bacteria — 30S subunit with IF1, IF3 binds mRNA where the Shine–Dalgarno sequence (AGGAGG) pairs with 16S rRNA; IF2-GTP brings fMet-tRNA to AUG in the P site; 50S joins (GTP hydrolysed) → 70S initiation complex. Eukaryotes — 40S with eIFs and Met-tRNA_i binds the 5′ cap and scans to the first AUG in a good context (Kozak sequence); 60S joins → 80S.
3. Elongation: (a) EF-Tu-GTP (eEF1A) brings the next aminoacyl-tRNA to the A site; (b) peptidyl transferase forms the peptide bond, transferring the chain to the A-site tRNA; (c) translocation by EF-G-GTP (eEF2) moves the ribosome one codon; deacylated tRNA moves to E site and leaves. The chain grows N→C terminus.
4. Termination: a stop codon in the A site is recognised by release factors (RF1: UAA, UAG; RF2: UAA, UGA; RF3-GTP — in eukaryotes eRF1 for all three + eRF3); peptidyl transferase hydrolyses the bond, the polypeptide is released and ribosome recycling factor (RRF) with EF-G separates the subunits.

Translation in chloroplasts and mitochondria: organelles have their own 70S-type ribosomes, tRNAs and genetic system resembling bacteria (endosymbiotic origin): start with fMet, use Shine–Dalgarno-like elements in many chloroplast mRNAs, sensitive to chloramphenicol. Most organelle proteins, however, are coded by nuclear genes, made on cytoplasmic 80S ribosomes and imported (e.g. RuBisCO small subunit via a transit peptide). Plant mitochondria use the universal code and import some tRNAs from the cytosol.

Protein modification

• De-formylation: in bacteria the formyl group of N-terminal fMet is removed by peptide deformylase soon after the chain emerges.
• Cleavage of terminal amino acids: methionine aminopeptidase removes the N-terminal Met (when the next residue is small); signal peptides and transit peptides are cut off by signal peptidases; pro-proteins are cut to active forms (e.g. proinsulin → insulin; zymogens).
• Protein splicing: an internal segment called an intein excises itself from a precursor protein and the flanking exteins are joined by a peptide bond — post-translational, self-catalysed (found in bacteria, archaea, yeast); used in biotechnology.
• Other modifications: phosphorylation, glycosylation, acetylation, methylation, lipidation, disulphide bond formation, ubiquitination (for degradation).

Protein folding — mechanism, role of chaperones, assembly

Anfinsen (RNase experiment) showed the amino-acid sequence contains the information for folding. Folding proceeds through rapid formation of secondary structures and a molten globule (hydrophobic collapse) to the native state of lowest free energy ("folding funnel"). In crowded cells chains may misfold or aggregate.

Molecular chaperones help proteins fold correctly without becoming part of the final structure:

• Hsp70 (DnaK in E. coli) binds hydrophobic stretches of nascent chains, preventing aggregation (ATP-dependent).
• Chaperonins (Hsp60): GroEL–GroES in bacteria (and Cpn60 in chloroplasts — the RuBisCO subunit binding protein): barrel-shaped chamber in which a protein folds in isolation, ATP-driven.
• Hsp90, small Hsps; protein disulphide isomerase and peptidyl-prolyl isomerase catalyse slow steps.

Assembly of proteins: subunits fold and then associate into multimers (quaternary structure), sometimes helped by assembly chaperones — e.g. RuBisCO L₈S₈ assembly in chloroplasts needs Cpn60 and assembly factors; misfolded proteins are degraded by proteasomes.

Likely exam questions

1. (Long, 8 marks) Describe the mechanism of protein synthesis (translation) in prokaryotes.
2. (Long, 8 marks) What is the genetic code? Explain its characteristics and the wobble hypothesis.
3. (Short, 4 marks) Compare 70S and 80S ribosomes.
4. (Short, 4 marks) Explain the role of chaperones in protein folding.
5. (Short, 4 marks) Describe the four levels of protein structure.

Syllabus topics (RTMNU)

• Bioinformatics: History, definition and scope; Bioinformatic tools for genetic studies
• Biological databases: Structure of a database; Types: Primary, secondary, composite, flat-file, relational, hierarchical; Database management system; database models – network, dimensional and object-oriented
• Sequences and nomenclature: IUPAC symbols; DNA and protein sequences; Directionality of sequences; Types of sequences: gDNA, cDNA, organellar DNA, Expressed sequence tag (EST)
• NCBI – the information resource: Functions; GenBank; Data retrieval tools (Entrez, PubMed, taxonomy browsers); BLAST
• Sequence alignment: Definition; Global and local alignment; Types: Pair-wise and multiple sequence alignment

Bioinformatics — history, definition and scope

Bioinformatics is the use of computers, statistics and information technology to collect, store, analyse and interpret biological data, especially DNA, RNA and protein sequences and structures. The term was coined by Paulien Hogeweg and Ben Hesper (1970).

History: Margaret Dayhoff's Atlas of Protein Sequence and Structure (1965) — first sequence database and the PAM matrices; Needleman–Wunsch global alignment (1970); Protein Data Bank (1971); Smith–Waterman local alignment (1981); GenBank (1982), EMBL (1980), DDBJ (1986); NCBI founded (1988); FASTA (1985) and BLAST (1990); Arabidopsis (2000) and human genome (2001–03) projects; next-generation sequencing (2005 onward).

Scope/applications: genome annotation and gene prediction, comparative genomics and phylogenetics, DNA barcoding, primer design, protein structure prediction, drug design (molecular docking), transcriptomics and proteomics data analysis, crop improvement (marker-assisted selection, genomic selection), systems biology.

Bioinformatic tools for genetic studies: BLAST and FASTA (similarity search), Clustal Omega/MUSCLE (multiple alignment), MEGA/PHYLIP (phylogenetic trees), Primer-BLAST (primer design), ORF Finder and gene prediction tools, ExPASy tools (ProtParam, translate), EMBOSS suite, Swiss-Model/AlphaFold (structure), RasMol/PyMOL (viewing).

Biological databases — structure and types

A database is an organised collection of data in records, each with fields (e.g. accession number, organism, sequence, references), stored so that it can be searched and retrieved. Each entry has a unique accession number.

• Primary databases: contain original experimental data submitted by researchers — nucleotide: GenBank (NCBI, USA), ENA/EMBL (Europe), DDBJ (Japan) — these three exchange data daily (INSDC); protein structure: PDB; protein sequence: UniProtKB/TrEMBL.
• Secondary databases: derived from analysis of primary data — curated or pattern information: UniProtKB/Swiss-Prot (curated), PROSITE (motifs), Pfam (protein families), PRINTS, InterPro, SCOP/CATH (structural classification).
• Composite databases: combine several primary databases with non-redundant entries — e.g. NCBI's NRDB (non-redundant), OWL.

By how data are stored:

• Flat-file database: data stored as plain text records one after another (e.g. GenBank flat file format with LOCUS, DEFINITION, ACCESSION, FEATURES, ORIGIN lines); simple but slow to search and hard to link.
• Relational database: data stored in related tables (rows and columns) linked by keys; queried with SQL (e.g. Ensembl uses MySQL).
• Hierarchical database: tree-like parent–child structure; each child has one parent (e.g. taxonomy tree, Gene Ontology-like organisation).

Database management system (DBMS): software that creates, stores, updates, retrieves and secures data and controls access — e.g. MySQL, Oracle, PostgreSQL.

Other database models: network model — records linked as a graph, a child may have many parents (many-to-many); dimensional model — fact tables with dimension tables (star schema) used in data warehouses for analysis; object-oriented model — data stored as objects with attributes and methods, suitable for complex biological objects (e.g. 3-D structures, pathways; AceDB).

Sequences and nomenclature

IUPAC symbols: nucleotides A, C, G, T (U in RNA); ambiguity codes R (A/G, purine), Y (C/T, pyrimidine), S (G/C), W (A/T), K (G/T), M (A/C), B (not A), D (not C), H (not G), V (not T), N (any base). Amino acids use one-letter codes (A Ala, R Arg, N Asn, D Asp, C Cys, Q Gln, E Glu, G Gly, H His, I Ile, L Leu, K Lys, M Met, F Phe, P Pro, S Ser, T Thr, W Trp, Y Tyr, V Val; X any, * stop).

Directionality: DNA/RNA sequences are written 5′ → 3′ (left to right); proteins from N-terminus → C-terminus. The complementary strand is written as the reverse complement.

Common format: FASTA — a header line beginning with ">" followed by the sequence lines.

Types of sequences:

• gDNA (genomic DNA): sequence as present in the genome — includes exons, introns, promoters and intergenic regions.
• cDNA (complementary DNA): DNA copied from mRNA by reverse transcriptase — only exons (coding sequence + UTRs), no introns; used to study expressed genes and to express eukaryotic genes in bacteria.
• Organellar DNA: chloroplast and mitochondrial genome sequences (e.g. rbcL, matK used in plant DNA barcoding).
• EST (Expressed Sequence Tag): a short (about 200–800 bp) single-pass sequence read from one end of a randomly picked cDNA clone; quick, cheap way to discover expressed genes, map genes and develop markers (EST-SSRs); stored in dbEST.

NCBI — the information resource

The National Center for Biotechnology Information (NCBI, 1988, part of the US National Library of Medicine, NIH, Bethesda) builds and maintains public databases and analysis tools.

• Functions: storing and distributing sequence, structure, literature and genome data; providing search and analysis tools (BLAST, ORF Finder, Primer-BLAST); research in computational biology; training and education.
• GenBank: the NIH genetic sequence database — an annotated collection of all publicly available nucleotide sequences, part of INSDC; each record has an accession number and version (e.g. AB123456.1).
• Entrez: the integrated search and retrieval system linking NCBI databases (Nucleotide, Protein, Gene, Genome, Structure, PubMed, Taxonomy etc.) — one query searches all, and records are cross-linked.
• PubMed: free search engine for biomedical and life-science literature (MEDLINE citations and abstracts), with links to full text (PubMed Central).
• Taxonomy browser: hierarchical classification and names of all organisms in NCBI databases; browse from kingdom to species and link to their sequences.
• BLAST (Basic Local Alignment Search Tool, Altschul et al., 1990): finds sequences in a database similar to a query using a fast heuristic local alignment. Programs: blastn (nucleotide vs nucleotide), blastp (protein vs protein), blastx (translated nucleotide query vs protein database), tblastn (protein query vs translated nucleotide database), tblastx. Output: score (bits), E-value (expected number of chance hits — smaller is more significant), % identity, query coverage and alignments.

Sequence alignment

Definition: arranging two or more sequences to identify regions of similarity that may reflect functional, structural or evolutionary relationships. Matches, mismatches and gaps (insertions/deletions, shown as "−") are scored using a scoring scheme (match/mismatch scores or substitution matrices like PAM and BLOSUM62, plus gap opening and extension penalties).

• Pairwise alignment: two sequences at a time (dot-plot, dynamic programming, BLAST 2 sequences). Used to find homology and percentage identity.
• Multiple sequence alignment (MSA): three or more sequences aligned together, usually by the progressive method (guide tree) — Clustal W/Clustal Omega, MUSCLE, T-Coffee, MAFFT. Reveals conserved regions/motifs, used to build phylogenetic trees and design primers.

Likely exam questions

1. (Long, 8 marks) What are biological databases? Classify them with examples of primary, secondary and composite databases.
2. (Long, 8 marks) Describe NCBI as an information resource with GenBank, Entrez, PubMed and BLAST.
3. (Short, 4 marks) Distinguish between global and local sequence alignment.
4. (Short, 4 marks) What is an EST? Give its uses.
5. (Short, 4 marks) Write the IUPAC symbols for nucleotides.