VIRUSES AND TRANSPOSONS

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VIRUSES AND TRANSPOSONS
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TRANSPOSONS

• Transposable elements
• Jumping genes
• Mobile DNA
• Able to move from one place to another within a
  cells genome
• Sometimes a copy is made and the copy moves
• Insertion requires target DNA sequences
• Many such sequences exist within the host genome

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TRANSPOSONS

• First discovered by Barbara McClintock in the
  1940s
• Kernel color in maize varied as sequences entered
  and exited pigment genes
• Complete inactivation ? no pigment
• Partial inactivation ? partial pigment
• Full excision ? normal pigment
• If this sounds crazy now, how do you think it
  sounded when a female scientist suggested it half
  a century ago?

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TRANSPOSONS

• Discovered them in 1940s/1950s, but nobody paid
  attention
• Ideas finally accepted in 1970s
• Nobel prize in 1983
• She was 81 years old!!

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TRANSPOSONS

• Insertion sequences
• Simplest bacterial transposons
• Consist solely of DNA necessary for the act of
  transposition
• Gene encoding transposase enzyme
• Short inverted repeats

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TRANSPOSONS

• Simple Transposition
• Target DNA sequences cut by transposase
• Transposon excised by transposase
• Ligation of transposon into target site
• DNA gaps filled by DNA polymerase

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TRANSPOSONS

• Replicative Transposition
• Transposon is replicated
• New copy inserted elsewhere into the genome
• Transposons increase in number

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TRANSPOSONS

• Retrotransposition
• Transposon is replicated through RNA intermediate
• Requires reverse transcriptase
• New copy inserted elsewhere into the genome
• Transposons increase in number

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TRANSPOSONS

• Composite transposons
• Longer and more complex
• Extra genes flanked by insertion sequences
• Formed when insertion sequences insert near each
  other

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TRANSPOSONS

• Composite transposons
• Extra genes may benefit the bacterium
• e.g., Antibiotic resistance genes
• Movement of antibiotic resistance genes is
  important in the formation of R plasmids

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R (RESISTANCE) PLASMIDS

• Carry genes conferring resistance to antibiotics
  or heavy metals
• Two regions
• R genes encode resistance
• May be multiple genes
• RTF codes for resistance transfer factor
• Pilus synthesis
• Origin of transfer
• Mobilization genes

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TRANSPOSONS

• Upon entering the genome of an organism,
  transposons can spread quickly
• P elements were introduced into Drosophila
  melanogaster in the 1950s
• Aside from some laboratory stocks, Drosophila
  worldwide now harbor P elements

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TRANSPOSONS

• Upon entering the genome of an organism,
  transposons can spread quickly
• P elements were introduced into Drosophila
  melanogaster in the 1950s
• Aside from some laboratory stocks, Drosophila
  worldwide now harbor P elements

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TRANSPOSONS

• Why do transposons exist?
• If they can damage genes, why doesnt natural
  selection weed them out of the gene pool?

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TRANSPOSONS

• Why do transposons exist?
• Selfish DNA Theory
• Genetic parasites conferring no selective
  advantage to the host cell
• Transposons possess characteristics allowing
  themselves to multiply within host cell DNA
• Proliferate without unduly damaging host

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TRANSPOSONS

• Why do transposons exist?
• Selective advantage?
• Increased genetic variation
• Advantages conferred by genes carried within
  transposons
• e.g., Antibiotic resistance genes
• e.g., Exon shuffling
• Transposons insert exons into coding sequences of
  genes
• Novel genes and proteins formed

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TRANSPOSONS

• Transposable elements have been found in
  bacteria, plants, fungi, and animals
• Probably occur in the genomes of all organisms
• Not always active

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TRANSPOSONS

• There are no currently active transposons in
  vertebrates
• Defective transposons can be found in vertebrate
  genomes
• By comparing DNA sequences of these defective
  transposons, a functional transposon can be
  reconstructed

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TRANSPOSONS

• Dr. Perry Hackett has reawakened a vertebrate
  transposon from a sleep of fifteen million years
• Sleeping Beauty
• University of Minnesota
• Discovery Genomics, Inc.
• http//www.discoverygenomics.net/index.phtml

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TRANSPOSONS

• Genes of interest can be inserted into the
  Sleeping Beauty transposon
• Engineered transposons can be used as an
  efficient means of delivering genes into
  vertebrate genomes
• Introduction of therapeutic genes
• Gene therapy

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VIRUSES
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UNIFYING FEATURES OF LIFE

• Composed of cell(s)
• Require energy
• Possess genetic material
• Reproduction
• Evolution (characteristic of populations)
• Etc.

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VIRUSES
?

• Share some of the unifying features of life
• Differ from normal life in some of these
  characteristics
• Are viruses alive?

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VIRUSES

• CELLULAR LIFE
• Composed of cell(s)
• Require energy
• Genetic material (DNA)
• Reproduction
• Evolution (populations)
• Etc.

• VIRUSES
• Not cellular
• Only when infecting
• DNA or RNA
• Only within host
• Very rapid evolution

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VIRUS STRUCTURE

• Simplest Viruses
• Naked viruses
• Naked nucleocapsid
• Genetic material
• DNA or RNA
• Single- or double-stranded
• Protein coat
• Capsid
• Surrounds nucleic acid

Adenovirus
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VIRAL GENOME

• DNA or RNA
• Single-stranded or double-stranded
• Linear or circular
• Single or multiple pieces
• Relatively small
• Capsid has finite size
• Packed very tight
• Up to 10X pressure of champagne bottle

• Relatively few genes
• Viruses 3 - hundreds
• E. coli 4,000
• H. sapiens 40,000
• Lacks genes for many essential functions
• Must borrow this machinery from host cells

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VIRAL CAPSID

• Composed of many identical protein subunits
• Capsomeres
• Determines viral shape
• Isometric / icosahedral
• Helical
• (Some more complex)
• Attachment proteins
• Spikes
• Attach virus to host cell
• Nucleic acid capsid nucleocapsid

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BACTERIOPHAGES

• Phage
• Virus infecting bacteria
• Most are naked viruses
• Most have complex shape

T4 bacteriophage
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VIRUS STRUCTURE

• Enveloped Viruses
• Genetic material
• Protein coat
• Envelope
• Phospholipid bilayer
• External to nucleocapsid
• Integral proteins
• Possesses attachment proteins
• Spikes
• Attach virus to host cell
• Matrix protein often inside envelope

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VIRUS STRUCTURE

• Additional Components
• Genetic material
• Protein coat
• Envelope
• Enzymes
• Present in some viruses
• Encoded by virus
• Required for viral life cycle
• Not available in host
• e.g., Integrase
• e.g., RNA-dependent RNA polymerase
• e.g., RNA-dependent DNA polymerase (reverse
  transcriptase)

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SIZE SIZE SIZE SIZE SIZE

• How small is small?
• Most microorganisms are small
• Viruses are among the smallest microorganisms
• 10 nm 500 nm range
• 100 1,000 times smaller than the cells they
  infect
• Frequently not removed by filter sterilization

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VIRAL REPLICATION CYCLE

• Viruses cannot reproduce themselves without
  assistance
• Lack machinery for energy harvesting, protein
  synthesis, etc.
• Must borrow this machinery from host cells
• Obligate intracellular parasites

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VIRAL REPLICATION CYCLE

• Viral reproduction requires infection of host
  cells
• Viral genetic material is introduced into host
  cell
• Host cell is reprogrammed
• Host cell produces many copies of the infecting
  virus

?
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VIRAL REPLICATION CYCLE

• Orderly series of steps
• Attachment (Adsorption)
• Penetration (Entry)
• Replication
• Assembly Maturation
• Release

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VIRAL REPLICATION CYCLE

• Attachment
• Adsorption
• Virus binds to host cell
• Viral surface proteins bind to cell surface
  receptors
• Different viruses require different receptors
• Different host cells possess different receptors
• Why would a cell possess receptors for a virus?

T4 bacteriophage
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VIRAL HOST RANGE

• The host range of a particular virus includes
  all cell types able to be infected
• These cells possess receptors to which the virus
  is specific
• e.g., Certain species
• e.g., Certain strains of a particular species
• e.g., Certain cell types within a multicellular
  organism

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VIRAL REPLICATION CYCLE

• Entry
• Penetration
• Viral genetic material enters cell
• Various mechanisms
• Portions of the virus may remain outside of host
  cell

T4 bacteriophage
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VIRAL REPLICATION CYCLE

• Production of Viral Components
• Various mechanisms
• Assembly Release
• Various mechanisms

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VIRUS - HOST INTERACTIONS

• Host metabolism completely taken over
• Lytic infection
• Host metabolism partially taken over
• (No specific name for this)
• Peaceful coexistence with host cell
• Lysogenic infection
• Latent infection
• Accomplished by temperate phages
• Generally involves the integration of the virus
  into the hosts DNA

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T4 LYTIC LIFE CYCLE
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l LYTIC VS. LYSOGENIC
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LYSOGENS

• Characteristics of cells possessing a provirus
• Immune to infections by same type of phage
• Proviral protein binds to inactivates infecting
  virus
• New genes ? new properties
• Corynebacterium diptheriae b prophage ?
  diptheria toxin ? diptheria
• Clostridium botulinum prophage ? toxin ?
  botulism
• Streptococcus pyogenes prophage ? toxin ?
  scarlet fever

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TRANSDUCTION

• Generalized transduction
• Bacterial DNA erroneously packaged inside capsid
• Infects new host
• Bacterial DNA transferred
• Any gene
• Facilitated by virulent (lytic) and temperate
  (lysogenic) phages

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TRANSDUCTION

• Specialized transduction
• Imperfect prophage excision removes some host DNA
• Packaged into capsid
• defective phage
• Infects new host
• Bacterial DNA transferred
• Genes near integration site
• Facilitated by temperate phages only

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ANIMAL VIRUS CLASSIFICATION

• Inherently difficult, changing
• Based mainly on
• Genome structure
• Virus particle structure
• Presence or absence of an envelope
• Reflects evolutionary relationships

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ANIMAL VIRUS CLASSIFICATION

• All viruses are not grouped into a single domain
  or kingdom
• There is no common ancestor for all viruses
• Viruses likely originated from fragments of
  mobile DNA
• Transposons or plasmids
• Essentially, escaped portions of host genomes
• Multiple origins of viruses

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ANIMAL VIRUS CLASSIFICATION

• Divided into families
• 14 RNA-virus families infect vertebrates
• 7 DNA-containing families infect vertebrates
• -viridae
• Members of families share common ancestor
• Relationship between families more complex
• Families further subdivided into genera

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ANIMAL VIRUS CLASSIFICATION

• Binomial nomenclature is not used in the naming
  of viruses
• Family (-viridae)
• Genus (-virus)
• Species
• Named for disease
• e.g., Polio ? poliovirus
• Types
• Akin to subspecies, strains, etc.
• Some types should be separate species

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HOST - VIRUS INTERACTIONS

• Bacteriophage host organism is single cell
• Possess rudimentary defense mechanisms
• Animal virus host organism is multicellular
• Possesses various defense mechanisms
• Immunity can exist
• Virus host coevolution
• Host ? more resistant
• Virus ? less pathogenic
• balanced pathogenicity
• Normal host is often asymptomatic
• Disease results when transmitted to a susceptible
  host
• e.g., Measles smallpox in New World indigenous
  populations

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VIRAL REPLICATION

• Replication of proteins
• Utilizes host cell ribosomes and other machinery
• Replication of nucleic acid
• Genetic material varies between families
• Methods of nucleic acidreplication variable
• NA replication often involves viral enzymes

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VIRAL REPLICATION

• Some of the enzymes required for viral
  replication have significant research uses
• e.g., Reverse transcriptase
• What else possesses this enzyme?

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VIRAL HOST RANGE

• Includes all cell types able to be infected
• e.g., Species, strains, cell types within a
  species
• These cells possess receptors recognized by virus
• Host range can be altered
• Phenotypic mixing
• Genetic reassortment

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PHENOTYPIC MIXING

• Animal cells sometimes simultaneously infected by
  two different viruses
• Host ranges overlap, but differ
• Viral genetic material and viral capsids
  mismatched
• Host range temporarily altered
• Can facilitate interspecies gene transfer

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GENETIC REASSORTMENT

• Some viruses possess segmented genomes
• e.g., Influenza virus
• Many strains of these viruses exist
• Host ranges overlap, but differ
• Two different strains can infect a single cell
• e.g., Bird and human viruses can both infect pigs
• RNA segments mixed and matched
• Antigenic shift
• New strain avoids immunity already in place

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PLANT VIRUSES

• Many plant diseases are caused by viruses
• Especially prevalent in perennial plants
• Yield can be severely reduced
• Without plants (food), we are dead
• Infection sometimes desirable
• e.g., Color variegation in tulips
• Have you seen anything like this before?