lecture 1-3 topic 1
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TOPIC 1
GENOME STUDIES
Dr Choo QC (TOPIC 1) 1
Genome
Genome is total genetic information possessed by anorganism in every cell, tissue, and organ in a body.
Every cells contain complete copy of instructions,written in the four-letter language of DNA (i.e. A, C,
T, G).
If the genome (DNA molecule) of a typical bacteriumis extended, it would be about 2mm in length. In
comparison, the diameter of the bacterium itself is
only about 0.001 mm.
Dr Choo QC (TOPIC 1) 2
Genome
The amount of protein sequence informationin a cell cannot be easily estimated from its
genome size because:
(a) Not all DNA codes for proteins- Introns
- Regulatory regions (e.g. promoters)
(b) Some genes exist in multiple copies
(c) The alternate splicing of the gene
Dr Choo QC (TOPIC 1) 3
Genome of Selected Organisms
Organism Number of Genes
(Approximate)
Number of base pairs
(x 106)
Escherichia coli 4500 4.6
Saccharomyces
cerevisiae
6000 12.1
Caenorhabditis
elegans
19000 95.5
Arabidopsis thaliana 25000 117
Drosophila
melanogaster
1300 180
Homo sapiens 25000 3200Dr Choo QC (TOPIC 1) 4
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Genomics
Genomics - study of genome
Involves large data sets (i.e. 3 billion base pairsfor human genome)
High-throughput methods (fast methods fordata collection)
Dr Choo QC (TOPIC 1) 5
Genomics
Genomics studies include:
DNA sequencing
Genomic library constructions
PCR amplification and cloning
Hybridization techniques
Dr Choo QC (TOPIC 1) 6
Genomics
Genome variation within a population,
Transcriptional control of genes,
Proteome (complete protein content of
a cell/organisms at a given time)
Dr Choo QC (TOPIC 1) 7
Histones (DNA
binding proteins)
Chromatin (DNA
histone complexes)
Nucleosome (8histone protein)
form core octamer
have linker
histones (act as
clamp prevent
coiled DNA from
detaching from
chromosome)
Dr Choo QC (TOPIC 1) 8
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Dr Choo QC (TOPIC 1) 9
Genes
An order sequence of nucleotides that encodes
a specific product
Physical and functional
units of heredity
Dr Choo QC (TOPIC 1) 10
Genes
May be turned on or off (by its regulatorymechanism) in response to the environment
e.g. Concentration of nutrients & stress
Development of the organism
Bacterial genomes may also have operons acontiguous of several genes to catalyze
successive steps of biochemical reactions
Dr Choo QC (TOPIC 1) 11
Genes
Genes comprise only about 2% of the human
genome
The remainder consists of non-coding regions,
Function: providing chromosomal structural
integrity and regulation - where, when, and in
what quantity proteins are made
Human genome is estimated to contain~25,000 genes
Dr Choo QC (TOPIC 1) 12
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09_25_Chromosome22.jpg
Dr Choo QC (TOPIC 1) 13
There are 23 Chapters, called CHROMOSOMES:
All the chapters being bind together called FOLDINGS
Each chapter contains several thousand stories, calledGENES
Each story is made up of paragraphs, called EXONS
which are interrupted by advertisements calledINTRONS
Each paragraph is made up of words, called CODONS
Each word is written in letters called BASES which isglued together with BONDS
And this is what that made up the GENOMEDr Choo QC (TOPIC 1) 14
Proteins
Large, complex molecules made up of chainsof small chemical compounds called amino
acids
Perform most life functions
Majority of cellular structures
Dr Choo QC (TOPIC 1) 15
Proteins
Nucleotide sequence can be translated intoamino acid sequence using the universal geneticcode
Chemical properties that distinguish the 20different amino acids
Cause the protein chains to fold up into specificthree-dimensional structures that define theirparticular functions in the cell
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Dr Choo QC (TOPIC 1) 17 18
Why Study Proteins?
Genomes Information
Proteins Action
Proteins rely on their regular three-dimensionalstructure for function. They have to have the rightshape and chemistry to carry out their biologicalrole
This means bringing together amino acids, notonly in a particular sequence, but also spatialrelationship
Dr Choo QC (TOPIC 1)
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Amino Acids
Monomeric building blocks of proteins Joined by covalent bond (peptide bond) Twenty different amino acids
- Same general structure- Differ in side chain (R group)- All organisms have same set of 20
Different activities and shapes of proteins due todifferent amino acid sequences
Dehydrationsynthesis
Carboxylgroup
Aminogroup
PEPTIDE BOND
Dr Choo QC (TOPIC 1) 20
What Amino Acids Look Like
C
R
H
N C
O
OH
H
H
side chain
amino
group
carboxyl
group
Different side-chain (R group)
Different chemical and physical propertiesDr Choo QC (TOPIC 1)
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Three Classes of Amino Acids
- Classification Based on Polarity(A) Nonpolar (hydrophobic)
G A V L I
M F W P
Dr Choo QC (TOPIC 1) 22
(B) Polar uncharged
(C) Charged : basic and acidic
S T C YN
Q
D EK R
H
Dr Choo QC (TOPIC 1)
THREE-DIMENSIONAL
STRUCTURES OFPROTEINS
23Dr Choo QC (TOPIC 1) 24
Primary Structure
Determines 2o, 3o, 4o structures
EXAMPLE: Sickle cell anemia - Single amino acidchange in hemoglobin related to disease
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GUG CAC CUG ACU CCU GAG GAG AAGval his leu thr pro GLU glu lys1 2 3 4 5 6 7 8
GUG CAC CUG ACU CCU GUG GAG AAGval his leu thr pro VAL glu lys1 2 3 4 5 6 7 8
Mutation (in DNA)
Normal mRNA
Normal protein
Mutant mRNA
Mutant protein
NOTE: Glu is a negatively charged amino acid and it is
replaced by Val, which has no charge
Sickle Cell Hemoglobin (HbS)
Dr Choo QC (TOPIC 1) 26
Sickle Cell Hemoglobin (HbS)
Caused by a point mutation(single A to T)
Amino acid substitution
(Glu to Val) in the 6th
position of -globins 146amino acid chain
Low oxygen conditionscause Hb to aggregate into
rod-shaped polymers This distort the shape of
RBC to a sickle shape
Dr Choo QC (TOPIC 1)
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Primary Structure
The amino acid sequence or polypeptide chain
Primary structure determines final shape and function
Secondary Structure
Repeated coiling or folding of the polypeptide by hydrogenbonding
Local description of structure
Major Types: -helix
-sheets
Loops & turns
Dr Choo QC (TOPIC 1) 28
(ii) (iii)
2o Structure
(i) -helix(ii) -sheet(iii) Loops and turns
(i)
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-Helix
Amino hydrogen (N-H) on nth residue bonds withcarbonyl oxygen (C=O) located 4 amino acidsaway (nth + 4)
A common secondary structure in both fibrous andglobular proteins
Side chain groups point outwards from helix
Amino acids with bulky side chains less commonin -helix
Glycine and proline destabilizes -helixDr Choo QC (TOPIC 1) 30
-Strand and -Sheet
Strands may be parallel / antiparallel Anti-parallel -sheets are more stable
Side chains point alternately above and below theplane of the beta-sheet
-sheet are common motifs in proteins
Dr Choo QC (TOPIC 1)
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Turns Loops with < 5 amino acids
are called turns Allows the peptide chain to
reverse direction Proline and glycine are
prevalent in -turns
Loops and turns= Non-repetitive structure
Loops Loops usually contain hydrophillic residues Connect -helices and -sheets
Dr Choo QC (TOPIC 1) 32
3o Structure
Third level of protein
organization
3-D arrangement
Types of tertiaryinteractions
(A) Bonds: Covalent Ionic
Hydrogen(B) Hydrophobic interactions
Hydrophobic
Interaction
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4o
Structure
Describes the organization ofsubunits in a protein withmultiple subunits
Subunits held together bynon-covalent interactions
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Dr Choo QC (TOPIC 1)
Proteins
Many new protein sequence data are nowbeing determined by translation of DNA
sequences, rather than by direct sequencing
of proteins (an expensive procedure)
However, one should remember that the
protein sequence translated from the genomesequence is a hypothetical structure until it is
verified experimentally
Dr Choo QC (TOPIC 1) 34
Proteomics
Proteome - complete set of proteins producedwithin a cell
Proteomics - the study of proteins
Proteome of an organism changes dependingon its environment stimulus (like heat shock,
growth)
Rate of synthesis of different proteins variesamong different tissues, different cell types
and state of activityDr Choo QC (TOPIC 1) 35
Picking out genes from genomes
Bioinformatics software can assist scientist infinding novel genes from genome
The software identifies open reading framesor ORFs - a region of DNA sequence that
begins with an initiation codon (ATG) and ends
with a stop codon
An ORF is a potential protein-coding regionDr Choo QC (TOPIC 1) 36
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Genome SequencingProjects
Dr Choo QC (TOPIC 1) 37
1977 First viral genome Sanger et al. sequence bacteriophage X174
This virus is 5386 base pairs (encoding 11 genes) Note: Accession J02482
1981 Human mitochondrial genome 16,500 base pairs
1986 Chloroplast genome
156,000 base pairs (most are 120 kb to 200 kb)
1995 First genome of a free-living organism, the bacterium
Hemophilus influenzaeDr Choo QC (TOPIC 1) 38
1997
More bacteria and archaea
Escherichia coli
4.6 Mb; 4200 proteins (38% of unknown function)
1998
First multicellular organism Nematode Caenorhabditis elegans
97 Mb; 19,000 genes.
1999
First human chromosome
Chromosome 22 (49 Mb, 673 genes)
Dr Choo QC (TOPIC 1) 39 Dr Choo QC (TOPIC 1) 40
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Significance and Importance
of Genome Studies
Dr Choo QC (TOPIC 1) 41
Potential benefits(A) Molecular medicine:
Improved diagnosis of disease - lead to more accuratediagnosis
Earlier detection of genetic predispositions to disease
Will be able to assess risk for certain diseases
e.g. cancer, Type II diabetes, heart disease
Drugs designed to target specific gene products thatcause disease
Gene therapy and control systems for drugs
Replacement of defective genes for certain diseases
Pharmacogenomics "custom drugs
Drug therapy based on genotype
Human Genome Project (and others)
Dr Choo QC (TOPIC 1) 42
(B) Archaeology, anthropology, evolution, andhuman migration
Study evolution through germline mutations in lineages
Study migration of different population groups based onfemale genetic inheritance
Study mutations on the Y chromosome to trace lineageand migration of males
Compare breakpoints in the evolution of mutations withages of populations and historical events
Human Genome Project (and others)
Dr Choo QC (TOPIC 1) 43
(C) DNA forensics (identification)
Identify potential suspects whose DNA may matchevidence left at crime scenes
Exonerate persons wrongly accused of crimes
Identify crime and catastrophe victims Establish paternity and other family relationships
Identify endangered and protected species as an aid towildlife officials (could be used for prosecuting poachers)
Detect bacteria and other organisms that may pollute air,water, soil, and food
Determine pedigree for seed or livestock breeds
Human Genome Project (and others)
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(D) Agriculture, livestock breeding, and bioprocessing Disease-, insect-, and drought-resistant crops
Healthier, more productive, disease-resistant farm animals
More nutritious produce
Biopesticides
Edible vaccines incorporated into food products
New environmental cleanup uses for plants
Human Genome Project (and others)
Dr Choo QC (TOPIC 1) 45
Genomes of Prokaryotes
Dr Choo QC (TOPIC 1) 46
Gene Regulation in Bacteria
Dr Choo QC (TOPIC 1) 47
The Operon
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The Operator
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Operons in E. coli
Dr Choo QC (TOPIC 1) 50
(a) Lac operon (Inducible Operon)
Dr Choo QC (TOPIC 1) 51
Lac operon (Inducible Operon)
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(a) Lactose absent, repressor active, operon off
Regulatorygene
Promoter
Operator
DNA lacZlacIDNA
mRNA
5
3
NoRNAmade
RNApolymerase
ActiverepressorProtein
Dr Choo QC (TOPIC 1) 53
(b) Lactose present, repressor inactive, operon on
lacI
lacoperon
lacZ lacY lacADNA
mRNA
5
3
Protein
mRNA 5
Inactive
repressor
RNA polymerase
Allolactose
(inducer)
-Galactosidase Permease Transacetylase
Dr Choo QC (TOPIC 1) 54
(b) Trp operon (Repressible Operon)
A repressible operon that is always on
Only turns off in the presence of its end
product, the amino acid tryptophan
Produces enzymes for production of tryptophan
Structural genes present within the tryptophanoperon code for repressible enzymes
Dr Choo QC (TOPIC 1) 55
(a) Tryptophan absent, repressor
inactive, operon on
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(b) Tryptophan present, repressor active, operon off
DNA
mRNA
Protein
Tryptophan(corepressor)
Activerepressor
No RNAmade
Dr Choo QC (TOPIC 1) 57
Inducible & Repressible Enzyme
Inducible enzymes usually function in catabolicpathways - synthesis is induced by a chemical
signal
Repressible enzymes usually function in anabolicpathways - synthesis is repressed by high levels of
the end-product
Regulation of the trp and lac operons involvesnegative control of genes because operons are
switched off by the active form of the repressor
Dr Choo QC (TOPIC 1) 58
Genome of prokaryotes
Large circular, double-stranded DNA
Usually < 5 Mbp
May contain plasmids
Environment-specific genes on plasmids andother types of mobile genetic elements
Dr Choo QC (TOPIC 1) 59
Genomes of prokaryotes
The protein-coding regions of bacterialgenomes:
Do not contain introns
Partially organized into operons
Genes that are located alongside one anothertranscribed into single mRNA molecule, under
common transcriptional control
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Genomes of prokaryotes
In bacteria, the genes of many operons codefor proteins with related functions
For instance, successive genes in the trpoperon of E. coli code for proteins that
catalyze successive steps in the biosynthesis of
tryptophan
Dr Choo QC (TOPIC 1) 61
Genome of
Escherichia coli
Dr Choo QC (TOPIC 1) 62
Genome ofEscherichia coli
Contains 4,639,221 bp in a single circular DNAmolecule, with no plastids
Relatively gene dense
Gene coding for proteins or structural RNAsoccupy ~89 % of the sequence
Average size of an ORF is 317 amino acids
Dr Choo QC (TOPIC 1) 63
Genome ofEscherichia coli
Most of the transcribe units contain only 1gene but E. colialso has operons where a setof genes grouped at one place
It is estimated that E. coli genome contains2584 operons
Operons vary in size, although few containmore than five genes
Genes within operons tend to have relatedfunctions
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Genome ofEschericia coli
The largest class of proteins is the enzymes approximately 30 % of total genes
Many enzymatic functions are shared by morethan one protein - arisen by duplication or
differ in specificity, regulation or intracellular
location
Dr Choo QC (TOPIC 1) 65
Genome ofEscherichia coli
4288 protein-coding genes
122 structural RNA genes
Non-coding repeat sequences
Regulatory elements
Transposable elements
Prophage remnants
Patches of unusual composition - likely to beforeign elements introduced by horizontal gene
transfer
Dr Choo QC (TOPIC 1) 66
Dr Choo QC (TOPIC 1) 67 Dr Choo QC (TOPIC 1) 68
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