Viral & Prokaryotic Genetics

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Viral Prokaryotic Genetics
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Introduction

• Viruses and prokaryotes are excellent model
  organisms
• Short generation times
• Small genomes
• Typically haploid
• What is a virus?
• Acellular
• Very small (1/10 or less of a bacteria)
• Composed of protein nucleic acid

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Viral Reproduction

• Obligate intracellular parasites
• Must use host cell for DNA replication, protein
  synthesis
• Outside host cell called protein
  capsid (coat) (DNA or
  RNA)
• Classified by
• Type of genome/nucleic acid
• Shape of virion (simple vs complex)
• Presence/absence of membrane
• Host cell lifecycle (viruses that infect
  bacteria bacteriophage)

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Viral Lifecycle

• Either lytic or lysogenic (burst now or burst
  later)
• Lytic cycle virus injects nucleic acid,
  reproduces, and kills host cell
• Lysogenic cycle virus injects nucleic acid,
  pauses, eventually re-activates kills host cell

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Lifecycle Cont

• Lytic cycle viruses that ONLY use this cycle are
  
• After infection, have early stage late stage
  transcription
• Early genes code for proteins that shut down
  normal cell transcription, eat host DNA,
  increase viral transcription, and stimulate
  transcription of late genes
• Late genes code for proteins that form the capsid
  and cause the host cell to (releasing
  all the newly made virions)

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Lifecycle Cont

• Lysogenic cycle viruses that use this and the
  lytic cycle are
• If a bacteria is infected with a temperate phage,
  becomes a lysogenic bacteria (has inactive viral
  DNA integrated into its genome known as
  )
• At some point prophage is activated, removes from
  chromosome and enters lytic cycle
• So, when cell is healthy, virus is dormant once
  damaged or stressed virus emerges and seeks out
  new hosts

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Animal Viruses

• Viruses are common in vertebrates but are highly
  variable
• Some use to transmit from one
  species to another, others are air-borne
• 3 methods of entering host cells
• If naked (no membrane envelope) taken up via
  endocytosis -gt breaking down vesicle membrane -gt
  break down capsid -gt release of viral nucleic acid

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Animal Viruses Cont

• 2. If membrane envelope present, use receptor
  proteins (glycoproteins) on membrane surface to
  be endocytosized into cell
• 3. Fuse viral and host cell membranes to release
  nucleic acid into cytoplasm
• (note enveloped viruses generally use host cell
  plasma membrane to form new envelope)

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Animal Viruses Cont

• Many viruses have RNA, nto DNA as their genetic
  material
• normal viruses (like influenza) have their own
  replication enzyme (RNA-dependant RNA polymerase)
  to make mRNA, which makes glycoproteins (for new
  envelopes) new viral RNA

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Animal Viruses Cont

• Retroviruses (like HIV) do more using
  
• , form complementary DNA (cDNA) strands
  (provirus) which integrates into host DNA until
  activated
• Then it turns on transcription of viral RNA,
  leading to formation of viral glycoproteins,
  capsids, and envelopes

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How Are Viral Genes Regulated?

• If temperate, must gauge health of host to
  determine lifecycle use a genetic switch
• Have 2 regulatory proteins compete for 2 promoter
  regions on phage DNA (one for each lifecycle)
• Each protein acts against the promoter (negative
  feedback)

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Prokaryotes

• Cellular (thus living) organisms that reproduce
  asexually
• Binary fission produces (genetically
  identical cells) via rapid cell division
  (generation time 20-30 min)

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How to Recombine Genes When Youre Asexual

• Conjugation exchange of bits of DNA between
  prokaryotic cells (forms recombinant genomes)
• Requires a contact mechanism (the sex
  ) that forms a larger
  through which small snips of linear DNA
  can pass
• Donor DNA then lines up with host DNA and
  cross-over can occur (can get up to half of
  transferred genes into host genome)

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How to Recombine Genes When Youre Asexual Cont

• picking up any old
  DNA you find and recombining it with your own
  genome (remember R and S strain pneumococci?)
• in a
  virus-infected bacteria, bits of bacterial DNA
  get packaged into virion capsids which then get
  injected into other bacterial cells

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Plasmids

• Small, circular chromosomes (fewer genes than the
  main chromosome) that can be exchanged during
  conjugation
• Can inherit an entire plasmid, since no
  recombination (cross over) is necessary
• Types of plasmids
• Metabolic factors code for ability to perform
  unusual metabolic functions (eat weird things)

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Plasmids Cont

• factors (F factors) have genes
  that allow conjugation (sex pilus conjugation
  tube genes)
• factors provide resistance
  to antibiotics or other negative conditions
• Antibacterial resistance is a growing problem, in
  part due to overuse

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Transposable Elements

• Segments of DNA that can be removed re-inserted
  into chromosomes
• Option one relatively short, self-replicating
  strands that randomly copy and insert into a
  chromosome
• Option two longer sequences cut (not copied) and
  re-inserted elsewhere (carry transposable
  elements plus one or more genes are called
  transposons)

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How Are Prokaryotic Genes Regulated?

• Not all proteins are needed all the time - save
  energy resources by not making them
• Regulatory mechanism must be
• Dependent upon environmental conditions
• Highly efficient
• How to limit protein?
• Downregulate transcription (less mRNA)
• Destroy mRNA prior to translation
• Prevent translation at the ribosome
• Destroy protein after translation
• Prevent protein from functioning

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Prokaryotic Regulation Cont

• If you want a protein, use substances called
  inducers (work on inducible proteins)
• Proteins that are always produced are
• proteins

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Regulation Cont
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Regulation Cont

• Structural genes code for primary structure of
  proteins (ie any genes that can be transcribed
  into mRNA) can act together
• Genes that code for enzymes that perform the same
  job (isozymes) may all be transcribed onto the
  same piece of mRNA if they share a promoter site
• So, one promoter can impact transcription of
  multiple (near by) genes
• How can you shut down transcription?

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Operons

• Transcription can be shut down in prokaryotes by
  putting up a roadblock between the promoter and
  the gene(s) it regulates
• The section between the promoter and gene(s) is
  called the if it binds to a
  transcription will not occur
• Taken together (promoter, operator, genes) called
  an

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Operons Cont

• A repressor binds to 2 thigns the operator and
  the inducer(s)
• If an inducer is present, it binds to the
  repressor, causing an (conformational)
  change that prevents it from binding to the the
  operator
• If the inducer is present, the repressor cannot
  function transcription occurs

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The lac operon

• Lactose is a possibel food source for E. coli
  that is not always present (it prefers glucose)
• If lactose is present, requires enzymes to
  metabolize if lactose is not present, dont need
  these enzymes
• Lactose is an inducer this is an inducible system

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Repressible Systems

• Unlike inducible systems where too many reactants
  are present, repressible systems prevent
  transcription when too much product is present
• Repressor must bind to
  (product) in order to bind to the operator
• So in inducible systems, binding to a reactant
  allows transcription in repressible systems,
  binding to a product prevents transcription
• Both of these are operator-repressor systems

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Promoter Efficiency

• In addition to allowing transcription, proteins
  can be upregulated by increasing promoter
  efficiency
• repression is a mechanism
  of regulation based on making RNA polymerase less
  tightly bound to DNA (less transcription)
• Regulatory protein (CRP) binds to cAMP, allowing
  RNA polymerase to bind better

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In Summary

• There are both positive (catabolite repression)
  and negative (inducible and repressible systems)
  control mechanisms that allow prokaryotic cells
  to regulate gene expression

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Genomics

• What do we gain from sequencing an organisms
  genome?
• Find the coding regions (open reading frames)
• Determine the amino acid sequence of specific
  proteins
• Find the regulatory sequences (promoters/terminat
  ors)

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Genomics

• genomics use genomic
  sequencing to establish the function of the genes
• Use annotation to find out what unknown
  proteins/genes code for
• genomics comparing the
  genomes of different organisms (determine
  relatedness, ecological function)