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ECE 6760: Modeling and Analysis of Biological

Networks

Chris J. Myers

Lecture 1: Brief Introduction to Biochemistry

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Systems Biology

• Systems biology is study of molecular systems, above single

genes and proteins, at the level of pathways.

• Enabled by new high throughput experimental techniques:

– cDNA microarrays and oligonucleotide chips

– Mass spectrometric identification of gel-separated protiens

– 2-hybrid systems

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Genetic Regulatory Networks

• Gene expression is regulated at many molecular levels.

• Regulation can occur at transcription, translational, or later.

• Involves numerous feedback loops.

• Some well understood at bacterial level (phage λ), situation

much more complex in higher organisms.

• Tension between stochastic and deterministic behavior.

• Role of noise and tradeoffs between robustness and evolvability

are important open questions.

• These networks are major focus of this course.

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Metabolic Networks

• Enzymatic processes to transform food into energy, and both

biosynthesis and biodegradation catalyzed by enzymes.

• Begins with key precursor metabolites (12 in E. Coli).

• Derive ∼100 building blocks (amino acids, nucleotides, etc.).

• Most biosynthetic reactions require ATP.

• Degradative reactions generate ATP.

• Typical reaction involves combinations of substrate with

enzyme to breakdown substrate or synthesize new product.

• Regulated by multiple processes.

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Protein Networks

• Communication and signaling networks with basic reactions

between two or more proteins.

• Involved in signal transduction cascades.

• Used to transfer information about the environment of the cell.

• Can be viewed as information processing networks.

• Protein circuits receive signals, process them, produce outputs

either as change of state or action (movement, secretion).

• Protein, metabolic, and regulatory networks overlap.

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Basic Method

• Identify all the players of the system.

• Perturb each component and record global response.

• Build a global model and new testable hypothesis

• Repeat.

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Analysis of Genetic Regulatory Systems

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Brief Introduction to Biochemistry

• Chemical reactions

• Macromolecules

• Genomes

• Cells and their structure

• Genetic circuits

• Metabolic networks

• Protein networks

• Cell replication

• Cell differentiation

• Viruses

• Bioinformatics and systems biology

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Atoms and Molecules

• All material created or destroyed via chemical reactions.

• Chemical reactions combine atoms to form molecules and

combine simpler molecules to form more complex ones.

• They also work in reverse.

• Atoms are the basic building block for all matter.

• 98% of living organisms consist of: hydrogen (H), carbon (C),

nitrogen (N), oxygen (O), phosphorus (P), and sulfur (S).

• Atoms form molecules via covalent, ionic, and hydrogen bonds.

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Chemical Reactions

2H2 + O2

k→ 2H2O

• H2, O2, and H2O are chemical (or molecular) species.

• Subscripts indicate H and O present in dimer form.

• The molecules H2 and O2 are known as the reactants.

• The water molecule is known as the product.

• 2’s indicate 2 H2 used to produce 2 water molecules.

• These numbers are known as the stoichiometry of the reaction.

• Since matter conserved, atom counts on each side must equal.

• Some reactions in this course may not have this property.

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Rate Constant

• k above arrow is the rate constant.

• It indicates probability or speed of this reaction.

• Used in many of the modeling techniques in this course.

• Often difficult to determine for bio-chemical reactions.

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Law of Mass Action

• Rate of a chemical reaction is governed by rate constant and

concentrations of reactants raised to power of stoichiometry.

• This is the law of mass action.

• The rate of water formation is:

d[H2O]

dt= 2k[H2]

2[O2]

where [H2O], [H2], and [O2] represent the concentration of

water, hydrogen dimers, and oxygen dimers.

• 2 in front of the k is due to fact that this reaction produces

two molecules of water.

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Laws of Thermodynamics

• Chemical reactions must obey the laws of thermodynamics.

• First law is that energy can be neither created or destroyed.

• Second law is entropy must increase.

• These two laws can be combined into a single equation:

∆H = ∆G + T∆S

where ∆H is change in bond energy, ∆G is change in free

energy, T is the absolute temp., and ∆S is change in entropy.

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Gibb’s Free Energy

• ∆G is also known as the Gibb’s free energy after J. Willard Gibbs

who introduced this concept in 1878.

• Consider a reversible reaction of the form:

A + BKeq↔ C + D

where Keq = k/k−1 is the equilibrium constant.

• The Gibb’s free energy for the forward reaction is:

∆G = ∆G◦ + RT ln{([C][D])/([A][B])}

where R = 1.987 cal/mol is gas constant and T is temperature.

• When negative, forward reaction can occur spontaneously.

• When positive, reverse reaction can occur spontaneously.

• When zero, the reaction is in a steady state.

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Gibb’s Free Energy (cont)

• The value of ∆G◦ is related to Keq:

∆G◦ = −RT ln Keq

• Combining equations results in:

∆G = RT lnk−1[C][D]

k[A][B]

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Hydrolysis of ATP

• How do chemical reactions with positive free energy occur?

• Free energies of chemical reactions are additive.

• Hydrolysis of ATP releases energy:

ATP + H2O ↔ HPO2−

4 + ADP.

• Coupling with other reactions allows them to occur.

• These types of ATP reactions occur in all living organisms.

• ATP is the universal energy currency of living organisms.

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Enzymes

• Activation energy barrier must be overcome.

• An enzyme, or catalyst, can accelerate a reaction without

being consumed by the reaction.

• Modifier is a species that is not consumed by a reaction.

• Often enzyme amount much smaller than other reactants.

• Enzymes do not effect free energy of the reaction, but only

help the reaction overcome its activation energy barrier.

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Michaelis-Menten Equation

• A basic enzymatic reaction:

E + Sk1

→

←

k2

ES k3

→ E + P

where enzyme, E, used to change substrate, S, into product, P .

• When [S] >> [Et], rate is often approximated using the

Michealis-Menten equation:

d[P ]

dt= Vmax

[S]

[S] + KM

where Vmax = k3[Et] and KM = (k2 + k3)/k1.

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Michaelis-Menten Derivation

• Derived using steady state assumption that [ES] reaches final

concentration quickly (i.e., d[ES]/dt ≈ 0).

• Using the law of mass action:

d[P ]

dt= k3[ES]

d[ES]

dt= k1[E][S]− k2[ES]− k3[ES]

• Using total concentration of enzyme, [Et]:

[E] = [Et]− [ES]

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Michaelis-Menten Derivation (cont)

• Using d[ES]/dt = 0 and above equations:

[ES] =[Et][S]

k2+k3

k1

+ [S]

• After substitution:

d[P ]

dt=

k3[Et][S]k2+k3

k1

+ [S]= Vmax

[S]

[S] + KM

• This is the quasi-steady state assumption.

• If k3 << k2, can be simplified even further:

d[P ]

dt=

k3[Et][S]k2

k1

+ [S](1)

• This is the rapid equilibrium assumption.

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Macromolecules

• Nearly 70 percent of a living organism made up of water.

• Remainder largely macromolecules of 1000s of atoms.

• There are four types:

– Carbohydrates

– Lipids

– Nucleic acids

– Proteins

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Carbohydrates

• Made up of carbon and water (Cn(H2O)m where m ≈ n).

• Often called sugars.

• An example is glucose.

• Important source of chemical energy.

• Powers nearly all processes of a cell.

• Also part of the backbone for DNA and RNA.

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Lipids

• Made up of mostly of carbon and hydrogen atoms.

• Primary use is to form membranes.

• Membranes separate cells from one another and create

compartments within cells as well as having other functions.

• Examples include fats, oils, and waxes.

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Nucleic Acids

• Store information within living organisms.

• Composed of base bound to sugar and phosphate molecule.

• Two forms: DNA (deoxyribonucleic acid) and RNA

(ribonucleic acid).

• Most organisms genetic information encoded in DNA

sequences, but a few viruses use RNA.

• A DNA strand (chain) is made up of four chemical bases:

adenine (A) and guanine (G), which are called purines, and

cytosine (C) and thymine (T), referred to as pyrimidines.

• Each base has slightly different composition of O, C, N , H.

• Sequence of nucleotides encode the instructions to construct

product produced by a gene.

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The Four DNA Bases

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Nucleic Acids (cont)

• A strand of DNA has a sense of direction, in that it is always

synthesized in the 5’ to 3’ direction.

• The so-called 5’ end terminates in a 5’ phosphate group

(-PO4); the 3’ end terminates in a 3’ hydroxyl group (-OH).

• DNA is a double-stranded molecule consisting of two chains

running in opposite directions.

• A-T and G-C base pairs are complementary.

• One strand of DNA acts as a template to direct the synthesis

of a complementary strand.

• Thus, DNA sequences are readily copied.

• Chemical makeup of this base pairing creates a force that

twists the DNA into its coiled double helix structure.

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Ribonucleic Acids

• Ribonucleic acid (RNA) is a chain, or polymer, of nucleotides

with the same 5’ to 3’ direction of its strands.

• Ribose sugar of RNA has a 2’ hydroxyl group, uracil replaces

thymine nucleotide, and is mostly single-stranded molecule.

• All genes that code for proteins are first made into an RNA

strand called a messenger RNA (mRNA).

• mRNA carries the information encoded in DNA to the protein

assembly machinery, or ribosome.

• The ribosome complex uses mRNA as a template to synthesize

the exact protein coded for by the gene.

• DNA also codes for ribosomal RNAs (rRNAs), transfer RNAs

(tRNAs), and small nuclear RNAs (snRNAs).

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Proteins

• Basic building blocks of nearly all the machinery of a cell.

• Each cell contains thousands of different proteins.

• Long chains with as many as 20 kinds of amino acids.

• Genetic code carried by DNA specifies order and number of

amino acids and, therefore, shape and function of the protein.

• Code from DNA is transferred to RNA through transcription.

• mRNA is translated by a ribosome into protein.

• mRNA decoded in blocks of three bases, or codons.

• Protein built one amino acid at a time, with order determined

by the order of the codons in the mRNA.

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The Genetic Code

U C A G

U UUU Phenylalanine UCU Serine UAU Tyrosine UGU Cysteine

UUC Phenylalanine UCC Serine UAC Tyrosine UGC Cysteine

UUA Leucine UCA Serine UAA Stop UGA Stop

UUG Leucine UCG Serine UAG Stop UGG Tryptophan

C CUU Leucine CCU Proline CAU Histidine CGU Arginine

CUC Leucine CCC Proline CAC Histidine CGC Arginine

CUA Leucine CCA Proline CAA Glutamine CGA Arginine

CUG Leucine CCG Proline CAG Glutamine CGG Arginine

A AUU Isoleucine ACU Threonine AAU Asparagine AGU Serineine

AUC Isoleucine ACC Threonine AAC Asparagine AGC Serineine

AUA Isoleucine ACA Threonine AAA Lysine AGA Arginine

AUG Methionine ACG Threonine AAG Lysine AGG Arginine

G GUU Valine GCU Alanine GAU Aspartate GGU Glycine

GUC Valine GCC Alanine GAC Aspartate GGC Glycine

GUA Valine GCA Alanine GAA Glutamate GGA Glycine

GUG Valine GCG Alanine GAG Glutamate GGG Glycine

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Proteins (cont)

• In 1961, Nirenberg and Matthaei correlated the first codon

(UUU) with amino acid phenylalanine.

• A given amino acid can have more than one codon.

• These redundant codons usually differ at the third position.

• Serine is encoded by UCU, UCC, UCA, and/or UCG.

• Redundancy is key to accommodating mutations that occur

naturally as DNA is replicated and new cells are produced.

• Some codons do not code for an amino acid at all but instruct

the ribosome when to stop adding new amino acids.

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Protein Structure

• Protein folds into a specific 3-dimensional configuration.

• Shape and position of the amino acids in this folded state

determines the function of the protein.

• Understanding and predicting protein folding is an important

area of research.

• The structure of a protein is described in four levels.

– Primary structure - sequence of amino acids.

– Secondary structure - patterns formed by amino acids that

are close (ex. α-helicies and β-pleated sheets).

– Ternary structure - arrangement of far apart amino acids.

– Quaternary structure - arrangement of proteins that are

composed of multiple amino acid chains.

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What is a Genome?

• All of the 30 million types of organisms use the same basic

materials and mechanisms to produce building blocks

necessary for life.

• Information encoded in the DNA within its genome is used to

produce RNA which produces proteins.

• A genome is divided into genes where each gene encodes the

information necessary for constructing a protein.

• Some also control the timing for when other genes should

produce their proteins.

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What are Genes?

• In 1909, Danish botanist Wilhelm Johanssen coined the word

gene for the hereditary unit found on a chromosome.

• 50 years earlier, Gregor Mendel characterized hereditary units

as factors–differences passed from parent to offspring.

• Mendel was an Austrian monk who experimented with his pea

plants in the monastery gardens.

• Normally they self-fertilize, but he manipulated their

parentage and thus their traits using a pair of clippers.

• Discovery went largely ignored for nearly 50 years until three

researchers essentially duplicated his experiments.

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How Many Genes Do Humans Have?

• In February 2001, two largely independent draft versions of the

human genome were published in Nature.

• Both estimated 30,000 to 40,000 genes in human genome

(today’s estimate is between 20,000 and 25,000).

• How do scientists estimate the number of genes in a genome?

– Open reading frames, a 100 bases without a stop codon;

– Start codons such as ATG;

– Specific sequences found at splice junctions; and

– Gene regulatory sequences.

• When complete mRNA sequences known, software can align

start and end sequences with the DNA sequence.

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What is Contained in Our Genome?

• Over 98% of genome has unknown function (“junk” DNA).

• Sequences that code for proteins are called structural genes.

• Regulatory sequences are start/end of genes, sites for initiating

replication/recombination, or sites to turn genes on/off.

• “Repetitive DNA”, short sequences repeated 100s of times,

make up 40 to 45 percent of our genome.

• Although have no role in the coding of proteins, they are an

excellent “marker” by which individuals can be identified.

• “Pseudogene” are believed to be a remnant of a real gene that

has suffered mutations and is no longer functional.

• Believed to carry a record of our evolutionary history.

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Introns and Exons

• Genes make up about 1% of the total DNA in our genome.

• Eukaryotic gene is not found in continuous stretch.

• The coding portions of a gene, called exons, are interrupted by

intervening sequences, called introns.

• Both exons and introns are transcribed into mRNA, but before

transported to ribosome, the mRNA transcript is edited.

• Removes introns, joins exons together, and adds unique

features to end of transcript to make a “mature” mRNA.

• It is still unclear what all the functions of introns are, but may

serve as the site for recombination.

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One Gene–One Protein?

• About 40 percent of the expressed genome is alternatively

spliced to produce multiple proteins from a single gene.

• This process may have evolved to limit effects of mutations.

• Genetic mutations occur randomly, and the effect of a small

number of mutations on a single gene may be minimal.

• However, an individual having many genes each with small

changes could weaken the individual, and thus the species.

• If single mutation affects several alternate transcripts, it is

likely individual may not survive.

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What is a Cell?

• The structural and functional unit of all living organisms.

• Some organisms, such as bacteria, are unicellular.

• Other organisms, such as humans, are multicellular.

• Humans have an estimated 100,000,000,000,000 cells!

• Each cell can take in nutrients, convert these into energy, carry

out specialized functions, and reproduce as necessary.

• Each cell stores its own set of instructions in its genome for

carrying out each of these activities.

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Prokaryotes and Eukaryotes

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Prokaryotic Organisms

• Life arose on earth about 4 billion years ago.

• The first types of cells were prokaryotic cells.

• They are unicellular organisms that lack a nuclear membrane.

• They do not develop or differentiate into multicellular forms.

• Bacteria are the best known and most studied form.

• Some bacteria grow in masses, but each cell is independent.

• They are capable of inhabiting almost every place on the earth.

• They lack any of the intracellular organelles and structures.

• Most of the functions of organelles are taken over by the

prokaryotic plasma membrane.

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Prokaryotic Architectural Features

• Prokaryotic cells have three architectural regions:

– Appendages called flagella and pili, proteins attached to

the cell surface;

– A cell envelope consisting of a capsule, a cell wall, and a

plasma membrane; and

– A cytoplasmic region that contains the cell genome (DNA)

and ribosomes and various sorts of inclusions.

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Eukaryotic Organisms

• They include fungi, mammals, birds, fish, invertebrates,

mushrooms, plants, and complex single-celled organisms.

• Eukaryotic cells are about 10 times the size of a prokaryote

and can be as much as 1000 times greater in volume.

• Have membrane-bounded compartments called organelles.

• Most important is the nucleus that houses the cell’s DNA.

• They use same genetic code and metabolic processes as

prokaryotes, but higher level of organizational complexity

permits truly multicellular organisms.

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The Plasma Membrane (A Cell’s Protective Coat)

• Outer lining of a eukaryotic cell.

• Separates and protects a cell from its environment.

• Made mostly of proteins and lipids.

• Embedded within are a variety of molecules that act as

channels and pumps, moving molecules into and out of the cell.

• In prokaryotes, usually referred to as the cell membrane.

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The Cytoskeleton (A Cell’s Scaffold)

• Acts to organize and maintain the cell’s shape.

• Anchors organelles in place.

• Helps during endocytosis, the uptake of external materials.

• Moves parts of the cell in processes of growth and motility.

• Involves many proteins each controlling a cell’s structure by

directing, bundling, and aligning filaments.

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The Cytoplasm (A Cell’s Inner Space)

• The large fluid-filled space inside the cell.

• In prokaryotes, this space is relatively free of compartments.

• In eukaryotes, “soup” within which organelles reside.

• Home of the cytoskeleton.

• Contains dissolved nutrients, helps break down waste products,

and moves material around the cell.

• Contains many salts and is an excellent conductor of electricity,

creating the perfect environment for the mechanics of the cell.

• The function of the cytoplasm, and the organelles which reside

in it, are critical for a cell’s survival.

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Organelles

• They are a set of “little organs” that are adapted and/or

specialized for carrying out one or more vital functions.

• Organelles are found only in eukaryotes and are always

surrounded by a protective membrane.

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The Nucleus: A Cell’s Center

• A spheroid membrane-bound region that contains genetic

information in long strands of DNA called chromosomes.

• Most conspicuous organelle found in a eukaryotic cell.

• Separated from the cytoplasm by nuclear envelope which

isolates and protects DNA from molecules that could damage

its structure or interfere with its processing.

• Where almost all DNA replication and RNA synthesis occurs.

• During processing, DNA is transcribed into mRNA.

• mRNA is transported out of the nucleus, where it is translated

into a specific protein molecule.

• In prokaryotes, DNA processing takes place in the cytoplasm.

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The Ribosome: The Protein Production Machine

• Found in both prokaryotes and eukaryotes.

• It is a large complex composed of RNAs and proteins.

• They process genetic instructions carried by mRNA.

• Translation is the process of converting a mRNA’s genetic code

into the exact sequence of amino acids that make up a protein.

• Protein synthesis is extremely important, so there are a large

number of ribosomes (100s or 1000s) in a cell.

• They float freely in the cytoplasm or sometimes bind to

another organelle called the endoplasmic reticulum.

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The Endoplasmic Reticulum and the Golgi

Apparatus: Macromolecule Managers

• The endoplasmic reticulum (ER) is the transport network for

molecules targeted for modifications and specific destinations.

• The ER has two forms: the rough ER and the smooth ER.

• The rough ER has ribosomes adhering to its outer surface.

• Translation of the mRNA for proteins that either stay in the

ER or are exported out of the cell occurs at these ribosomes.

• The smooth ER serves as the recipient for those proteins

synthesized in the rough ER.

• Proteins to be exported are passed to the Golgi apparatus for

further processing, packaging, and transport to other locations.

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Mitochondria and Chloroplasts

• Mitochondria are self-replicating organelles that occur in

various numbers, shapes, and sizes.

• Have two functionally distinct membrane systems:

– The outer membrane, which surrounds the organelle; and

– The inner membrane, which has folds called cristae that

project inwards to increase its surface area.

• Plays a critical role in generating energy in the eukaryotic cell.

• Chloroplasts are similar but are found only in plants.

• Convert sun’s light energy into ATP using photosynthesis.

• Both surronded by a double membrane, have their own DNA,

and are involved in energy metabolism.

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Organelle Genome

• Plants and animals have genome within some organelles.

• Mitochondrial DNA is only inherited from our mother.

• Independent aerobic function may have evolved from bacteria

living inside other organisms in a symbiotic, relationship.

• These organisms evolved to become incorporated into the cell.

• Many diseases caused by mutations in mitochondrial DNA.

• Mitochondrial Theory of Aging suggests accumulation of

mutations in mitochondria drives aging process.

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Lysosomes and Peroxisomes

• Often referred to as the garbage disposal system of a cell.

• Spherical, bound by membrane, and rich in digestive enzymes.

• Peroxisomes often resemble a lysosome, but are self replicating,

whereas lysosomes are formed in the Golgi complex.

• Lysosomes contain three dozen enzymes for degrading proteins,

nucleic acids, and certain sugars called polysaccharides.

• These enzymes work best at low pH, reducing risk that they

will digest their own cell if they escape from the lysosome.

• The cell could not house such destructive enzymes if they were

not contained in a membrane-bound system.

• Lysosome can digest foreign bacteria that invade a cell.

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Lysosomes and Peroxisomes (cont)

• They also recycle receptor proteins and other membrane

components and degrade worn out organelles.

• They can even help repair damage to the plasma membrane by

serving as a membrane patch, sealing the wound.

• Peroxisomes rid body of toxic substances, such as hydrogen

peroxide, and contain enzymes for oxygen utilization.

• High numbers of peroxisomes can be found in the liver.

• All enzymes in them are imported from cytosol and have a

special sequence, called a PTS or peroxisomal targeting signal.

• They also have membrane proteins for importing proteins into

their interiors and to segregate into daughter cells.

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Genetic Circuits

• Genes encoded in DNA used as templates to synthesize mRNA

through the process of transcription.

• Genes include coding sequences and regulatory sequences.

• Regulatory sequences can bind to other proteins which in turn

either activate or repress transcription.

• Transcription is also regulated through post-transcriptional

modifications, DNA folding, and other feedback mechanisms.

• This regulatory network increases an organism’s complexity.

• Behavior analogous to electrical circuits in which multiple

inputs are processed to determine multiple outputs.

• Therefore, these regulatory networks known as genetic circuits.

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An Overview of Transcription and Translation

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Transcription

• Initiated at promoter site by RNA polymerase (RNAP).

• Promoter is a unidirectional sequence found on one strand

which instructs RNAP where to start and in which direction.

• RNAP unwinds double helix at that point and begins synthesis

of an mRNA complementary to one of the strands of DNA.

• This strand is called the antisense or template strand, whereas

the other strand is referred to as the sense or coding strand.

• Synthesis proceeds in a unidirectional manner.

• Terminates when polymerase stumbles upon a stop signal.

• In eukaryotes, not fully understood, but prokaryotes have short

region of G’s and C’s that folds in on itself causing polymerase

to trip and release the nascent, or newly formed, mRNA.

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Regulation of Transcription

• Ability of RNAP to bind to promoter site can be either

enhanced or precluded by transcription factors.

• They recognize portions of the DNA sequence near the

promoter region known as operator sites.

• Those that help RNAP bind are activators and those that

block RNAP from binding are repressors.

• These sequences can be cis-acting (affecting adjacent genes),

or trans-acting (affecting distant genes).

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Methylation

• Transcription also regulated by variations in DNA structure.

• One chemical modification of DNA, called methylation,

involves the addition of a methyl group (-CH3).

• Methylation frequently occurs at cytosine residues preceded by

guanine bases, often in vicinity of promoter sequences.

• Inhibits transcription by attracting a protein that binds to

methylated DNA, interfering with polymerase binding.

• The methylation status of DNA often correlates with its

functional activity.

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Translation

• Ribosome has two subunits.

• Small subunit finds mRNA to begin translating.

• Large subunit has two sites for amino acids to bind.

• A site accepts transfer RNA (tRNA) bearing an amino acid.

• P site binds the tRNA to the growing chain.

• Each tRNA has a specific acceptor site that binds a particular

triplet of nucleotides, called a codon,

• Also has an anti-codon site that binds a sequence of three

unpaired nucleotides, the anti-codon, which binds to the codon.

• Also has specific charger protein that only binds to a specific

tRNA and attaches correct amino acid to the acceptor site.

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Translation (cont)

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Translation (cont)

• Start signal is the codon ATG that codes for methionine.

• A tRNA charged with methionine binds to the start signal.

• Large subunit binds to the mRNA and the small subunit, and

so begins elongation, the formation of the polypeptide chain.

• After the first charged tRNA appears in the A site, the

ribosome shifts so that the tRNA is now in the P site.

• New tRNAs, corresponding to codons of the mRNA, enter the

A site, and a bond is formed between the two amino acids.

• The first tRNA is now released, and the ribosome shifts again

so that a tRNA carrying two amino acids is now in the P site.

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Translation (cont)

• This continues until the ribosome reaches a stop codon.

• Ribosome breaks apart releasing the mRNA and new protein.

• Note that a protein will often undergo further modification,

called post-translational modification.

• Translational regulation occurs through the binding of

repressor proteins to a sequence found on an RNA molecule.

• Translational control plays a significant role in the process of

embryonic development and cell differentiation.

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Metabolic Networks

• The energy conversion pathways of a cell.

• Complex sugars broken down into glucose which enters the cell

through glucose transporters in the cell’s membrane.

• Metabolic networks break down glucose to make ATP using:

– Glycolysis

– Pyruvate oxidation

– Citric acid (Kreb’s) cycle

• Products of each pathway are reactants in following pathway.

• Glycolysis occurs in the cytoplasm while all the later steps

occur in the mitochondria.

• Capable of generating enough ATP to run all the cell functions.

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Glycolysis

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Glycolysis

• Requires no oxygen and is referred to as anaerobic metabolism.

• Glycolysis occurs in the cytoplasm outside the mitochondria.

• Glucose is broken down into a molecule called pyruvate.

• Each reaction produces hydrogen ions to make ATP.

• Only four ATP molecules can be made from one of glucose.

• In prokaryotes, glycolysis is the only method to convert energy.

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Pyruvate Oxidation

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Pyruvate Oxidation

• Begins with two molecules of pyruvic acid.

• Next, pyruvic acid is altered by the removal of a carbon and

two oxygens, which go on to form carbon dioxide.

• When CO2 is removed, energy is given off, and NAD+

molecule is converted into the higher energy form NADH.

• Another molecule, coenzyme A, then attaches to the remaining

acetyl unit, forming acetyl CoA.

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Citric Acid (Kreb’s) Cycle

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Citric Acid (Kreb’s) Cycle (cont)

• Acetyl CoA binds to a four-carbon molecule called oxaloacetate

to make a six-carbon molecule called citric acid.

• Citric acid is then broken down and modified in a stepwise

fashion releasing hydrogen ions and carbon molecules.

• The carbon molecules are used to make more carbon dioxide.

• The hydrogen ions are picked up by NAD and another

molecule called flavin-adenine dinucleotide (FAD).

• Eventually, the process produces the four-carbon oxaloacetate

again, ending up where it started off.

• Further processing of the ions released can result in 24 to 28

ATP molecules from one molecule of glucose.

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Protein Networks

• A sequence of biochemical reactions used by a cell to convert

one type of signal or stimulus into another type.

• Also known as signal transduction networks or signaling

cascades.

• Used to respond to a variety of stimuli from their environment

ranging from temperature and light changes to the presence of

chemicals in its neighborhood.

• Responses range from activation of a gene or metabolic

network to the cell moving or modifying its shape.

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Protein Networks (cont)

• Usually started by binding of an extracellular signaling

molecule to a receptor on the cell membrane.

• A receptor is typically only sensitive to a very specific

signaling molecule, or ligand.

• Ligand usually only exerts influence via the receptor since

often cannot pass through the cell membrane.

• This binding triggers a change in shape to the receptor which

in turn triggers events to occur within the cell.

• Second messengers are produced to pass the message conveyed

by the stimulus within the cell.

• Variety of second messengers: G protein, cyclic AMP, calcium

ions, and protein kinases and phosphatases.

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Receptor Tyrosine Kinase (RTK) Pathway

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Receptor Tyrosine Kinase (RTK) Pathway

• Involved in a number of processes including growth, migration,

survival, and differentiation.

• Initiated by the binding of a peptide such as epidermal growth

factor (EGF) to the RTK receptor.

• Extracellular ligand-binding domain is connected by a helix through

membrane to tyrosine kinase within the cytoplasm.

• This activates tyrosine kinase by autophosphorylation.

• Phosphorylation is the addition of a phosphate (PO4) to a enzyme

to activate or deactivate it.

• This phosphorylation produces binding sites for proteins such as

GRB2 bound to the SOS protein.

• This activates SOS which activates Ras which stimulates MAP

kinase which activates a variety transcription factors.

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Cell Communication

• A major use of protein networks is cell communication.

• Bacteria may form colonies of cells that communicate by

secreting signaling molecules that are monitored by other cells.

• In quorum sensing, bacteria sense quantities of a signaling

molecule in the medium secreted by all cells in a colony.

• When level exceeds a threshold, the bacteria know a quorum is

reached and can take on a more aggressive lifestyle.

• In multicellular organisms, protein networks used by cells to

send messages to each other.

• Signaling networks also used during organism development to

guide cell differentiation.

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Cell Replication

• Fundamental property of living things is ability to reproduce.

• All cells arise from pre-existing cells, their genetic material

must be replicated and passed from parent cell to progeny.

• All multicellular organisms inherit their genetic information

specifying structure and function from their parents.

• Prokaryotes reproduce through binary fission.

• Eukaryotes replace damaged or worn out cells through a

similar process called mitosis.

• To reproduce, eukaryotes use meiosis to create cells called

gametes that fuse to form the beginning of a new organism.

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Binary Fission

• For most unicellular organisms, reproduction is a simple

matter of cell duplication, also known as replication.

• They replicate all of their parts and then split into two cells - a

type of asexual reproduction called binary fission.

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An Overview of DNA Replication

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DNA Replication

• First, helicase unwinds a portion of the parental DNA.

• Next, DNA polymerase binds to one strand of the DNA.

• DNA polymerase begins to move along the DNA strand in the

3’ to 5’ direction, using the single-stranded DNA as a template.

• This leading strand is necessary for forming new nucleotides

and reforming a double helix.

• Since synthesis only occurs in 5’ to 3’ direction, a second DNA

polymerase is used to bind to the other template strand.

• This molecule synthesizes discontinuous segments of

polynucleotides, called Okazaki fragments.

• Another enzyme, called DNA ligase, is responsible for stitching

these fragments together into what is called the lagging strand.

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Variations

• Theoretically results in two identical cells, but bacterial DNA

has a relatively high mutation rate.

• This makes them capable of developing resistance to antibiotics

and helps them invade into a wide range of environments.

• They also have mechanisms for exchanging genetic material.

– Conjunction: direct joining of two bacteria, which allows

their circular DNAs to undergo recombination.

– Transformation: absorb remnants of DNA from dead

bacteria and integrate into their own DNA.

– Transduction: genes are transported into and out of cell by

bacteriophages or by plasmids.

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Overview of Mitosis

• Nucleus is divided to produce two identical daughter nuclei.

– Interphase: chromosomes replicate to produce sister

chromatids.

– Prophase: chromosomes are condensed, nuclear envelope

breaks down, and a large protein network, spindle, attaches

to each sister chromatid.

– Metaphase: chromosomes aligned perpendicular to spindle.

– Anaphase: molecular motors pull the chromosomes away

from the metaphase plate to the spindle poles of the cell.

– Telophase: cell divides, nuclear envelope reforms, and the

chromosomes relax and decondense.

• The cell can now replicate its DNA again during interphase

and go through mitosis once more.

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Overview of Mitosis

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Cell Cycle Control and Cancer

• During cell cycle, surveillance system monitors cell for DNA

damage and failure to perform critical processes.

• If this system senses a problem, a network of signaling

molecules instructs the cell to stop dividing.

• These “checkpoints” let the cell know whether to repair the

damage or initiate programmed cell death, apoptosis.

• Apoptosis ensures damaged cell is not further propogated.

• Protein p53 accepts signals of DNA damage and stimulates

production of inhibitory proteins to halt DNA replication.

• Without proper p53 function, DNA damage accumulates

unchecked and damaged gene progresses into a cancerous state.

• Defects in p53 are associated with a variety of cancers.

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Meiosis

• Producing a whole new organism requires sexual reproduction.

• To reproduce, a process called meiosis produces gametes.

• Meiosis reduces the chromosome number in half.

• In the second step, the sperm and egg join to make a single

cell, which restores the chromosome number.

• This joined cell then divides and differentiates into different

cell types that eventually form an entire functioning organism.

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Overview of Meiosis I

• Meiosis I, reduction division, reduces chromosome number from

diploid (two copies) to haploid (one copy).

• Meiotic division only occurs in cells associated with sex organs.

• Prophase I : virtually identical to prophase in mitosis.

• Metaphase I : pairs aligned on either side of metaphase plate.

• Chromatid arms may overlap and fuse, resulting in crossovers.

• Anaphase I : spindle fibers contract, pulling the homologous pairs

away from each other and toward each pole of the cell.

• Telophase I : cleavage furrow forms but nuclear membrane is not

reformed and chromosomes do not disappear.

• At the end of Telophase I, each daughter cell has a single set of

chromosomes, half the total number in the original cell.

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Overview of Meiosis

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Overview of Meiosis II

• Meiosis II is a mitotic division of each haploid cell.

• Prophase II : new set of spindle fibers form and the chromosomes

begin to move toward the equator of the cell.

• Metaphase II : all chromosomes align with the metaphase plate.

• Anaphase II : the centromeres split, and the spindle fibers shorten,

drawing the chromosomes toward each pole of the cell.

• Telophase II : a cleavage furrow develops, nuclear membrane is

formed, and the chromosomes fade.

• After Meiosis II, there will be a total of four daughter cells, each

with half the number of chromosomes as original cell.

• For males, all four cells will develop into sperm cells.

• For females, 3 cells abort, only 1 develops into an egg cell.

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Recombination–The Physical Exchange of DNA

• All organisms suffer a small number of mutations, or random

changes in a DNA sequence, during DNA replication.

• Recombination is large-scale rearrangement of a DNA molecule.

• Position of gene on a chromosome is called a locus.

• Two different versions of a gene, or alleles, may appear at a

given locus.

• During Meiosis I, the two strands of a chromosome pair may

physically cross over one another.

• This may cause strands to break apart at crossover point and

reconnect to the other chromosome, resulting in an exchange.

• Recombination results in a new arrangement of maternal and

paternal alleles on the same chromosome.

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Recombination

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Recombination (cont)

• Although genes appear in same order, the alleles are different.

• Explains why offspring from same parents look so different.

• Is possible to have any combination of parental alleles.

• This theory of “independent assortment” of alleles is

fundamental to genetic inheritance, but there is an exception.

• Recombination influenced by proximity of one gene to another.

• If two genes are located close together on a chromosome, the

likelihood of separation of these two genes is less.

• Linkage describes the tendency of genes to be inherited

together as a result of their location on the same chromosome.

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Linkage Disequilibrium

• Situation in which some combinations of genes or genetic

markers occur more or less frequently than expected from their

distance apart.

• Scientists apply this concept when searching for a gene that

may cause a particular disease.

• They do this by comparing the occurrence of a specific DNA

sequence with the appearance of a disease.

• When a high correlation between the two, they know they are

getting closer to finding the appropriate gene sequence.

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DNA Repair Mechanisms

• Maintaining accuracy of DNA genetic code is critical for both

the long- and short-term survival of cells and species.

• Sometimes, normal activities introduce mutations while others

are caused by exposure to chemicals, radiation, etc.

• Positive changes result in different version of a gene that is

beneficial in face of a new disease or environmental condition.

• Negative ones cause disease by preventing a vital protein from

being made or encode a defective protein.

• Counteracted by a vigorous surveillance and repair system.

• Number of enzymes capable of repairing damage to DNA.

• These systems also differ in the degree to which they are able

to restore the normal, or wild-type, sequence.

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Categories of DNA Repair Systems

• Photoreactivation: genetic damage caused by UV radiation is

reversed by illumination with visible or near-UV light.

• Nucleotide excision repair : is used to fix DNA lesions, such as

single-stranded breaks or damaged bases.

• Recombination repair : fixes DNA damage by a strand

exchange from the other daughter chromosome.

• Base excision repair : allows for the identification and removal

of wrong bases, typically attributable to deamination.

• Mismatch repair : recognizes inappropriately matched bases in

DNA and replaces one of the two bases.

• SOS repair : is process that occurs in bacteria including

mutagenesis or cell elongation without cell division.

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Cell Differentiation

• Not all cells are alike, skin cells are certainly different from

cells that make up our inner organs.

• All cell types in our body are all derived from a single,

fertilized egg cell through differentiation.

• Differentiation is process by which an unspecialized cell

becomes specialized.

• During differentiation, certain genes are turned on, or become

activated, while other genes are switched off, or inactivated.

• This process is intricately regulated.

• As a result, a differentiated cell will develop specific structures

and perform certain functions.

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Mammalian Cell Types

• Three basic categories of cells make up the mammalian body:

germ cells, somatic cells, and stem cells.

• Nearly all of the approximately 100,000,000,000,000 cells in an

adult human has its own copy of the genome.

• The majority of these cells are diploid.

• Somatic cells include most of the cells that make up our body,

such as skin and muscle cells.

• Germ cells are any that give rise to gametes–eggs and

sperm–and are continuous through the generations.

• Stem cells have the ability to divide for indefinite periods and

to give rise to specialized cells.

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Human Development

• Begins when a sperm fertilizes an egg and creates a single cell.

• After fertilization, this cell divides into identical cells.

• About 4 days after fertilization, these cells begin to specialize,

forming a hollow sphere of cells, called a blastocyst.

• Blastocyst has an outer layer of cells, and inside this hollow

sphere, there is a cluster of cells called the inner cell mass.

• Inner cell mass cells are referred to as pluripotent, that is, they

can give rise to many types of cells but not a whole organism.

• They undergo further specialization until they are committed

to give rise to cells that have a particular function.

• These more specialized stem cells are multipotent–capable of

giving rise to several kinds of cells, tissues, or structures.

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Differentiation of Human Tissues

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Where Do Viruses Fit?

• They are not classified as cells and therefore are neither

unicellular nor multicellular organisms.

• They are not “living” because they lack a metabolic system

and are dependent on host cells they infect to reproduce.

• They have genomes of either DNA or RNA which are either

double-stranded or single-stranded.

• Their genomes code for both proteins to package its genetic

material and those needed to reproduce.

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Viral Reproduction

• Because viruses are acellular, they must utilize the machinery

and metabolism of a host cell to reproduce.

• For this reason are called obligate intracellular parasites.

• Before entering host, it is a virion–package of genetic material.

• They can be passed from host to host either through direct

contact or through a vector, or carrier.

• Bacteriophages attach to the cell wall surface, make a small

hole, and inject their DNA into the cell.

• Others (such as HIV) enter the host via endocytosis, the

process in which a cell takes in material from its environment.

• After entering cell, its genetic material takes over cell and

forces it to produce new viruses.

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Types of Viruses

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Viral Reproduction (cont)

• The form of genetic material contained in the viral capsid

determines the exact replication process.

• If it has DNA, it is replicated by host along with its own DNA.

• If it has RNA, it is copied using RNA replicase making a

template to produce 100s of duplicates of the original RNA.

• Retroviruses use the enzyme reverse transcriptase to

synthesize a complementary strand of DNA which is then

replicated using the host cell machinery.

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Steps Associated with Viral Reproduction

1. Attachment, sometimes called absorption: The virus attaches

to receptors on the host cell wall.

2. Penetration: Viral genome moves through plasma membrane

into host, capsid of a phage remains outside.

3. Replication: Virus induces host to synthesize the necessary

components for its replication.

4. Assembly : The newly synthesized viral components are

assembled into new viruses.

5. Release: Assembled viruses are released from the cell and can

now infect other cells, and the process begins again.

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Viral Reproduction (cont)

• When the virus takes over cell, it directs host to manufacture

the proteins necessary for virus reproduction.

• The host produces three kinds of proteins:

– Early proteins, enzymes used in nucleic acid replication;

– Late proteins, proteins used to construct the virus coat; and

– Lytic proteins, enzymes used to break open the cell for exit.

• Viral product is assembled spontaneously, that is, the parts are

made separately by the host and are joined together by chance.

• Self-assembly often aided by molecular chaperones, or proteins

made by the host that help the capsid parts come together.

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Viral Reproduction (cont)

• New viruses leave the cell either by exocytosis or by lysis.

• Animal viruses instruct host’s endoplasmic reticulum to make

glycoproteins which collect in clumps along the cell membrane.

• Virus then discharged at these exit sites.

• Bacteriophages must break open, or lyse, the cell to exit.

• They have a gene that codes for an enzyme called lysozyme.

• This breaks down cell wall, causing the cell to swell and burst.

• New viruses released into the environment, killing the host.

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Why Study Viruses?

• They provide simple systems that can be used to manipulate

and investigate the functions of many cell types.

• They provide fundamental information about aspects of cell

biology and metabolism.

• Bacterial viruses have simplified study of bacterial genetics.

• Numerous studies demonstrate the utility of animal viruses as

probes for investigating different activities of eukaryotic cells.

• Provide important models of DNA replication, transcription,

RNA processing, and protein transport.

• This course uses a virus, phage λ, as a running example.

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Experimental Advancments

• Advances in high-throughput data acquisition techniques such

as microarrays and mass spectroscopy have led to an explosion

in the amount of biological data being generated.

• DNA microarrays are chips made of glass, plastic, or silicon

with an array of thousands of single-stranded DNA probes.

• When strands of mRNA extracted from cells during an

experiment are hybridized to complementary probes, they

flouresce.

• Used to measure gene expression levels which are correlated

with the levels of mRNA present.

• Also used to measure protein levels in a similar fashion.

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Microarrays

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Experimental Advancments (cont)

• Mass spectroscopy is a method in which ions are separated by

their mass-to-charge ratio.

• Since mass-to-charge ratio for different proteins is unique, used

to identify the presence of proteins during an experiment.

• These high-throughput experimental methods make it an

absolute requirement for data standards, computerized

databases, and the development of new computational tools.

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Bioinformatics

• Biology is being transformed from being a purely lab-based

science to also an information science.

• Biologists have had to draw assistance from those in

mathematics, computer science, and engineering.

• Result was fields of bioinformatics and computational biology.

• Major goal is to extract new biological insights from large and

noisy sets of data generated by high throughput technologies.

• Must create and maintain databases with massive amounts of

DNA sequence data.

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Bioinformatics (cont)

• Also involves developing efficient interfaces for researchers to

access, submit, and revise data.

• Has evolved to development of software that also analyzes and

interprets these types of data.

• In this course, we use the term bioinformatics to refer to the

analysis of static data such as sequence analysis of DNA and

protein sequences, techniques for finding genes or evolutionary

patterns, and cluster analysis of microarray data.

• Bioinformatics algorithms are not covered in this course.

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Systems Biology

• The focus of this course is the modeling and analysis methods

being developed for systems biology.

• Goal is to analyze high throughput data to develop models to

better understand how biological systems function.

• Concerned with the analysis of dynamic models.

• Only recently has this become possible.

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Systems Biology (cont)

• Ultimate goal is to develop methods which can give reasonable

predictions of experimental results.

• While it will never replace experimental methods, may help

experimentalists make better use of their time.

• Also may gain insight into mechanisms used by these biological

processes which may not be obtained by experiments.

• Eventually, may be possible that they could have substantial

impact on our society such as aiding in drug discovery.

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Standard Data Formats: SBML

• Standards for sequence data were absolutely essential.

• For systems biology, standard data formats being developed.

• One is the systems biology markup language (SBML).

• XML-based language to represent chemical reaction networks.

• All networks described in this lecture can be reduced to a set of

bio-chemical reactions.

• SBML model consits of a list of the chemical species and their

chemical reactions.

• Reaction includes a list of reactants, products, and modifiers.

• Also includes a mathematical description of the kinetic rate law

governing the dynamics of this reaction.

• SBML is ugly, but GUIs have been developed.

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Biological Databases

• Another essential item in the genomic-age was the

development of biological databases.

• These provide repositories for storing large bodies of data that

can be easily updated, queried, and retrieved.

• Databases store many things ranging from nucleotide sequences

within GenBank to biomedical literature at PubMed.

• Recently, a database for SBML models has been started.

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Tools

• Last essential piece is tools for analysis.

• Excellent list of bioinformatics tools at the NCBI website.

• List of systems biology tools that support SBML can be found

at at the SBML website.

• The remainder of this course concentrates on describing the

methods used by tools being developed for systems biology for

modeling and analysis.

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References

• NCBI’s Science Primer - http://www.ncbi.nlm.nih.gov

• Berg/Tymoczko/Stryer - “Biochemistry”

• Tozeren/Byers - “New Biology for Eng. and Comp. Scientists”

• Gonick/Wheelis - “The Cartoon Guide to Genetics”

• King/Stansfield - “A Dictionary of Genetics”

• Wikipedia - http://en.wikipedia.org

• PubMed and bioinformatics tools

(http://www.ncbi.nlm.nih.gov)

• SBML and systems biology tools (http://sbml.org/index.psp)

• SBML database - http://www.ebi.ac.uk/biomodels/

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