1. Define gene, chromosome, genetic code
    • Gene: region of DNA that codes for a protein
    • Chromosome: physical structure that holds genes
  2. Discuss the flow of genetic information (detals about replication, transcription, and translation)
    • Replication (DNA -> DNA)
    • DNA polymerase copies DNA (template read 5'->3', nucleotides are added to 3' -OH end)
    • Semi-conservative
    • Initiated by an RNA primer
    • Leading strand vs. lagging strand
    • DNA polymerase digests RNA primer, replaces with DNA
    • DNA ligase joins the discontinuous fragments (lagging strand)
    • Transcription (DNA -> RNA)
    • RNA polymerase binds to promoter sequence
    • DNA strand read 3'->5', mRNA synthesized 5'->3'
    • Stops at terminator sequence
    • RNA and RNA polymerase are released, double helix reforms
    • In eukaryotes introns must be removed before translation
    • Translation (RNA -> protein)
    • mRNA is translated in codons
    • start codon AUG
    • nonsense condons (end) UAA, UAG, UGA
    • 20 AA for 64 possible codons (61 sense, 3 nonsense)
    • Ribosomal subunits come together
    • tRNA moves through the EPA sites in <--- direction (A->P->E)
    • tRNA brings amino acids which are added to a chain
    • Polypeptide released on stop codon
    • Ribosomal components disassemble
  3. Regulation of bacterial gene expression by induction and repression
    • Induction: genes are expressed only when needed (eg. the Lac operon)
    • Repression: Genes are expressed until they are turned off (eg. Trp operon)
  4. Jacob, Monod, Griffiths
    • Jacob and Monod: responsible for the operon model of genes
    • Griffith: performed capsule/no capsule mouse experiment
  5. Structure and regulation of the Lac operon
    • I - P - O - Z - Y - A
    • Promoter (P): Where RNA polymerase binds
    • Operator (O): Where repessor will bind
    • ZYA: structural genes that code for 3 peptides.  All or nothing translation.
    • Regulatory gene (I): Encoding information for repressor
    • Inducible operon: off until turned on (active repressor bound to O when lactose is absent)
    • Lactose inactivates the repressor, causes transcription to occur.
    • Full expression of the lac operon requires both presence of lactose and absence of glucose (absence of glucose means cAMP high)
  6. Structure and regulation of the Trp operon
    • I - P - O - E - D - C - B - A
    • Promoter (P): Where RNA polymerase binds
    • Operator (O): Where repessor will bind
    • EDCBA: structural genes that code for 5 peptides.  All or nothing translation.
    • Regulatory gene (I): Encoding information for repressor
    • Repressible operon: on until turned off (Inactive repressor when Trp levels are low)
    • Trp activates the repressor (corepressor), halts transcription
  7. List and define the types of mutations, and explain their effects
    • Subsitutions: point mutations
    • Missense mutation: resuts in a changed A.A.
    • Nonsense mutation: results in a nonsense codon (ends translation)
    • Insertions/deletions: frame-shift mutations
    • Frameshift mutation: usually the most severe type
  8. List and explain how the different mutagens work
    • Mutagens: agent that causes mutations
    • Chemicals
    • Nitrous acid: oxidizes adenine
    • Radiation
    • Ionizing (X-Rays, gamma rays): cause formation of ions that can react with nucleotides
    • Non-ionizing (UV): tends to cause more mutations (thymine diamers)
  9. List and describe the two repair mechanisms of UV-damaged DNA.
    • Light-repair: photolyases separate thymine diamers
    • Nucleotide excision repair: endonuclease cuts the DNA and an exonuclease removes the damaged DNA.  DNA polymerase fills the gap.
  10. Define mutation rate and spontaneous mutation rate
    • Spontaneous mutation rate: 1 in 10^9 replicated base pairs, 1 in 10^6 replicated genes
    • Mutation rate: mutagens increase to 1 in 10^5 or 1 in 10^3 per replicated gene
  11. Describe the Ames test and its use
    • Two cultures are prepared of Salmonella that have lost the abilitity to synthesize histidine. 
    • The suspected mutagen is added to experimental sample
    • Each is poured onto a plate w/o histidine, incubated
    • Only bacteria whose phenotype has reverted to histidine-synthesizing will grow into colonies
  12. Describe how mutants are identified (two methods for selection)
    • Positive (direct) selection: detects mutant cells because they grow/appear differently.  Plate bacteria on a medium where the mutant, but not the parent, will grow. [Ames]
    • Negative (indirect) selection: detects mutant cells because they cannot perform a certain function.  [Replica plating]
  13. Describe the different methods of genetic transfer
    • Transformation: genes are transferred as naked DNA (picked up from environment)
    • Recombination:
    • Conjugation: genes are transferred across a pilus (protein bridge) from one cell to another (F- to F+)
    • Tranduction: genes are transferred by a bacteriophage
  14. Generalized vs specialized transduction
    • Generalized: phage infects the donor bacterial cell.  Phage DNA/proteins are made, bacterial chromosome is broken into pieces.  Occasionally during phae assembly pieces of bacterial DNA are packaged in a phage capsid.  Recombination occurs with new cell infection.
    • Specialized: Virus hides in chromosome.  When they excise themselves they can take part of bacterial chromosome with them.
  15. Define plasmid and describe its function + types of plasmids
    • Plasmid: extrachromosomal DNA that is self replicating
    • Useful for genetic engineering
    • 1. Conjugative plasmid (F factor): carries genes for sex pili and plasmid transfer
    • 2. Dissimilation plasmid: encodes enzymes for catabolism of unusual compounds
    • 3. R factors: encode antibiotic resistance
  16. Define transposon and explain the information they contain
    • Transponsons: segments of DNA that can move from one region of DNA to another
    • Insertion sequence: contains recognition sequences and transposase for cutting and resealing DNA
    • Complex transposons: carry other genes
    • Can cause resistances to antibiotics
  17. Discuss hybridization: colony, souther, western, northern
    • Hybridization: heating to separate strands, then allowing them to join with complementary strands
    • Colony: using a DNA proble to identify a cloned gene of interest (labeled probes are complementary to gene of interest)
    • Southern: used to detect specific DNA using a complementary DNA probe (eg Salmonella)
    • Western: proteins separated by electrophoresis can be detected by their reactions with antibodies (looking for RNA)
    • Northern: Looking for RNA with a complementary DNA probe
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