Applications of Molecular Biology – Lecture 1 Part 2 : Human Genome Project & Genome Sequencing

Genome Sequence & Structure – Basic Definitions

• Measuring DNA length
  • bp = base pair(s)
  • kb ( = kbp) = kilo-base pairs = $1\,000\text{ bp}$
  • Mb = mega-base pairs = $1\,000\,000\text{ bp}$
  • Gb = giga-base pairs = $1\,000\,000\,000\text{ bp}$
  • nt = number of nucleotides (used for ssDNA or RNA)

Chromosomes – Copy Number & Terminology

• Gamete carries one complete copy of the genome → haploid
• Diploid organism inherits one haploid set from each parent
  • Humans: $23$ chromosomes per haploid set → $2$ copies of every gene except on X & Y
• Euploid = cell with a normal complement of chromosomes
• Aneuploid = abnormal complement (e.g. trisomy)

Chromosome Anatomy

• Centromere
  • Constricted region; attachment site for mitotic spindle
• Telomere
  • Terminal DNA–protein structures; required for replication & stability
• Arms
  • p arm = short arm; q arm = long arm

Genomes & Cells

• Every organism possesses a unique genome encompassing all of its DNA
• All somatic cells of one individual harbour identical genomic DNA
• Differential gene expression → determines cell-specific functions (e.g. neuron vs. myocyte)

Genes, Chromosomes & DNA – Numbers to Remember

• Gene = DNA region controlling a hereditary trait; defined nucleotide sequence
• Human genome
  • ≈ $30\,000$ genes distributed on $23$ pairs of chromosomes
  • ≈ $3\times10^{9}$ bp (3 Gb)

Canonical Human Gene Architecture

• 5′ → 3′ orientation (DNA)
  • Upstream enhancers, promoter (with TATA box), 5′ UTR
  • Multiple exons separated by introns
  • 3′ UTR followed by poly-A signal (poly-A tail added post-transcriptionally)
• Primary transcript acquires 5′ cap & 3′ poly-A → mature mRNA exported to cytoplasm

Linear Arrangement of Genes on Chromosomes

• Genes occupy discrete, linear loci
• Example (Human Chr 22)
  • Two dsDNA molecules each $4.8\times10^{7}$ bp
  • Progressive magnification: chromosome → heterochromatic band → gene cluster → single gene (exon/intron structure)

Genome, Transcriptome & Proteome – Definitions

• Genome = complete DNA sequence
• Transcriptome = all RNA species expressed under given conditions (dynamic)
• Proteome = total protein complement at a point in time (dynamic)

Human Genome Project (HGP) 1990-2003 – Objectives

• Identify all ~$20\,500$ human genes
• Determine full 3 Gb sequence
• Store data in publicly accessible databases
• Develop/improve analytical tools & transfer technology to private sector
• Address ethical, legal & social implications (ELSI)

Technologies Spawned by HGP

• High-throughput, cost-effective sequencing platforms
• Software for variant detection & genome annotation
• Genomic databases + community distribution pipelines

Sequencing Strategies

• Shotgun sequencing (TIGR)
  • Randomly shear DNA (≈$1.5$ kb fragments), clone, sequence
  • Automated capillary electrophoresis
  • Assemble contigs via sequence overlap using bioinformatics
• Workflow summary
  1. Isolate chromosome or whole genomic DNA
  2. Shear → clone into sequencing vectors
  3. Sequence reads
  4. Computational assembly → contiguous genomic sequence

Milestones of the HGP

• 1994 – Genetic linkage map (2–5 cM resolution)
• 1995 – 50 % of physical map (100 kb resolution)
• Jun 2000 – Draft genome sequence announced
• Feb 2001 – First genome publication
• Output: $22.1$ billion raw bases ⇒ $3.9$ Gb assembled, ≈7-fold coverage

Key Insights from the HGP

• Total size = $3.1647$ Gb
• Average gene length ≈$3\,000$ bp; largest gene ≈$2.4$ Mb
• Gene count estimate = $30\,000$–$35\,000$
• >$50\%$ of genes: function still unknown (2001 estimate)
• <$2\%$ of genome encodes protein
• Repeats occupy ≈$50\%$ of genome
• GC rich segments correlate with gene-rich regions; AT rich with gene-poor
• Genes cluster non-uniformly; large intergenic deserts exist

Potential Applications of Genome Sequencing

• Molecular medicine
  • Precise diagnostics, early risk detection, gene therapy, personalised therapeutics
• Forensic science
  • Crime scene identification, exoneration, disaster victim ID, paternity testing, wildlife/species verification, pedigree analysis

Model Organism Genome Statistics (Representative)

• Human: $3.2$ Gb, $\approx19\,042$ protein-coding genes
• Mouse: $2.6$ Gb, $20\,210$ genes
• Rice: $389$ Mb, $37\,544$ genes
• Yeast $\left(S. cerevisiae\right)$: $12.1$ Mb, $6\,607$ genes
• E. coli: $4.6$ Mb, $3\,200$ genes
• HIV: $9.1$ kb, $9$ genes
  • Highlights disparity between genome size & gene number across taxa

Comparative Genomics

• Multi-species alignment of vertebrate genomes pinpoints highly conserved regions indicative of functional constraint

Genome Size vs. Complexity – C-Value Paradox

• Minimal genome size tends to rise with organismal complexity yet the correlation is weak (e.g. some amphibians > human genome)
• C-value = total DNA per haploid nucleus
• Paradox: genome size not proportional to coding capacity or morphological sophistication

How Many Genes Per Genome? Examples

• Range: $\approx470$ (Mycoplasma genitalium) → $\approx30\,000$ (rice, human)
• Not all genes essential; lethal loci counts often far lower (e.g. yeast $\sim1\,090$ essential)

Sequence Classes in the Human Genome

• Unique (single-copy) DNA
• Moderately repetitive DNA
• Highly repetitive (simple sequence repeats)
• Transposable elements
  • LINEs, SINEs, LTR elements, DNA transposons
• Repeats dispersed (tandem or inverted)

Quantitative Composition of the Human Genome (≈3.1 Gb)

• Genes & gene-related: $1.2$ Gb
  • Exons: $48$ Mb (<<2 %)
  • Introns + UTRs + gene fragments: $\approx1.152$ Gb
• Intergenic DNA: $1.0$ Gb
  • Interspersed repeats: $\approx400$ Mb (LINEs 640 Mb; SINEs 420 Mb; LTR 250 Mb; DNA transposons 90 Mb)
  • Other intergenic: $600$ Mb (microsatellites 90 Mb; various 510 Mb)

Future Challenge – Assigning Function to All Genes

• Even Drosophila: function known for only ≈$50\%$ of genes
• Key functional categories needing elucidation: signalling, cytoskeleton, cell cycle, transporters, unknown proteins

Recent Advances in Human Genome Sequencing (Illustrative Timeline)

• 1994 – Complete 2–5 cM linkage map
• 2001 – First genome assembly published
• 2007 – First individual (J. Craig Venter) genome sequenced
• 2008 – First African genome (Tishkoff et al.)
• 2010 – First European female genome
• 2010 – First Korean genome
• Continuing trend: celebrity & population genomes, large cohorts; costs falling dramatically

References for Further Study

• Alberts et al. "Essential Cell Biology" (5th ed., 2018) – Chs 1 & 10
• Lewin "Essential Genes" (4th ed., 2021) – Ch 5