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Define gene, chromosome, genetic code
- Gene: region of DNA that codes for a protein
- Chromosome: physical structure that holds genes
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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
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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)
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Jacob, Monod, Griffiths
- Jacob and Monod: responsible for the operon model of genes
- Griffith: performed capsule/no capsule mouse experiment
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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)
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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
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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
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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)
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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.
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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
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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
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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]
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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
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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.
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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
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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
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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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