lecture 1-3 topic 1

7/28/2019 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.


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


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)


Genomics

Genomics studies include:

DNA sequencing

Genomic library constructions

PCR amplification and cloning

Hybridization techniques


Genomics

Genome variation within a population,

Transcriptional control of genes,

Proteome (complete protein content of

a cell/organisms at a given time)


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)



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Genes

An order sequence of nucleotides that encodes

a specific product

Physical and functional

units of heredity


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


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



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09_25_Chromosome22.jpg


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


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


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


(B) Polar uncharged

(C) Charged : basic and acidic

S T C YN

Q

D EK R

H


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)


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


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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


(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


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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


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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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


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


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


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)


(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



(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



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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



Genomes of Prokaryotes


Gene Regulation in Bacteria


The Operon


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The Operator


Operons in E. coli


(a) Lac operon (Inducible Operon)


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


(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


(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


(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


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


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


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


Genome of

Escherichia coli


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



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



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) 67 Dr Choo QC (TOPIC 1) 68

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