bio120 lecppts week 10 11
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Bacterial Genetics
Review- Eukaryotic
1. DNA structure2. RNA structure
3. DNA replication
4. RNA transcription5. Protein translation
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Genome-all the genes present in a cell or virus;
procaryotes normally have one set of genes (haploid)eucaryotic microbes usually have two sets (diploid)
Genotype-the specific set of genes an organism possesses
Phenotype-the collection of characteristics of an organismthat an investigator can observe
Vertical transmission-transmission of genetic information
from parent to offspring
Horizontal transmission-genetic information transferred from
one cell to another
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1928- Griffith
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Avery, MacLeod, and McCarty
DNA is the genetic material
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tRNA
rRNA
codon
ribosomes
Vertical transmission
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Figure 8-4
Genome Transcription and Translation
Archaea ArchaeaEukarya Eukarya
Bacteria Bacteria
1
4
6 9 10
11 12 1315
1718
195
168
3 14
4
2 7
7 8
11
12 15 143
619
1716
915
2 10
13 18
4 TATA box and BRE sequencein promoter
1 Chromosome circular
versus linear2 Single chromosomeversus multiple
chromosomes3 Introns rare4Archaeal type introns
5 Inteins6 Histones7 DNA gyrase8 Reverse gyrase9 Multiple chromosomal
origins10 Eukaryotic origin
recognition complex
11 Eukaryotic type helicase
19 Telomeres and telomerase
12 B family DNA polymeraseis major replicative
enzyme13 Eukaryotic type sliding
clamp14 Restriction enzymes
15 RNAi16 Genome of double
stranded DNA17 Multiple retroelements
in genome
18 Centromeres19 18S, 28S and 5.8S rRNA
1 RNA used as a geneticmessenger
2 Polycistronic mRNA3 Cap and tail on mRNA
6 Multiple RNA polymerases
5 Repressors binding directly to
DNA in promoter
7 RNA polymerase II with 8 ormore subunits
8 Multiple transcription factorsneeded
9 Ribosomes synthesize proteins1070S versus 80S ribosomes
11 Ribosomal RNA sequencehomologies
12 Ribosomal proteinsequence homologies
13 Shine Dalgarno sequences14 Multiple translation factors15 Elongation factor sensitive to
diphtheria toxin16 N-formyl methionine versus
methionine17 tmRNA rescues stalled
ribosomes18 16S and 23S rRNA
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Table 7-1
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Minimal genome (480 proteins)
Bacterial chromosomes- 517kB-9400kB
Archaeal chromosomes-935kB-6500kB
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Fig.7-10
Parental strand
New strand
Semiconservative
replication
Initiation
Elongation
Termination
OriC
- origin of replication
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Transcription
RNA polymerase and promoters
mRNA-encode proteins
rRNA- ribosomes
tRNA-bind amino acids
sRNA-regulate gene expression, have catalytic activity,
or function as a combination of tRNA and mRNA
Transcription and translation in prokaryotes are coupled.
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OperonsDNA control
sequence
Structural gene-functional RNA molecule
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Eukaryotes
90% of DNA may be noncoding
Prokaryotes
Less that 15% DNA may be noncoding
Splicing
Remove introns
TailingAdenylic acid is added to the 3 end to
produce a polyA sequence about 200
nucleotides long (polyA tail)
Capping
7-methylguanosine is added to the 5
end (5' cap)
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Translation
Start codons AUG GUG UUG CUG
Stop codons UAA UAG UGA
Prokaryotes
30S
50S
Eukaryotes
40S
80S
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Extrachromosomal elements
1. Replicate autonomously within cells
2. Transferred between cells
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Table 11-1
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Plasmids as vectors for cloning
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Genome sequencing
-bacterial and archaeal genomes
-analysis of these genome sequences
ORFs function based on biochemical or genetic studies
large number of hypothetical ORFs with no known function
.
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3D - protein sequence has a 3D model built by homology, or a known 3D structure.
clear_function - protein sequence has functional annotation derived from probable homologues.
tentative_function - protein sequence has functional annotation derived from tentative
homologues
homologue - protein sequence has probable homologues of unknown function
no_homologue - protein sequence has no probable homologues.
E. coli
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Week 11
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In the experiment which proved that DNA was the genetic
material, the method of
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How do bacteria exchange genetic material?
1. Transformation-donor DNA in the environment is taken up by the bacteria
2. Transduction-donor DNA uptake is mediated by a virus
3. Conjugation-transfer involves cell-to-cell contact and a conjugative plasmid
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1. Transformationcompetence-a cell that is able to take up a molecule of DNA and be transformed
artificial competence-induction of competence by treating bacteria with certaincompounds under specific temperature or other factors
electroporation-treating cells with pulsed electric fields to open small pores in themembranes
transfection-transformation with DNA from virus
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Why do species undergo natural transformation?
a. Scrounge DNA from dead cells
b. Use DNA released from dead cells to repair own damaged genomes
c. Adjust to new environment by acquiring new genes
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Gram-positiveStreptococcus pneumoniae
translocasomea. Binding proteinb. Transmembrane
porea. Nuclease
Gram-negative- Must pass through outer membrane
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Conjugative plasmideg. F (fertility) plasmid (F factor)-transferable-circular DNA (99 159 bp)-contain genes needed for pilus formation and DNA export-can integrate into chromosome of host: Episomes
Curing-elimination of plasmids from host cellsConjugation- cell-to cell transfer of plasmids
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Sex pilus-acts as recognition molecule to locate sensitive cell (plasmid minus).-when + and - cell are attached by pilus, cells pulled together
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Conjugation (mating)
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Hfr strains
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Bacterial pheromones-chemicals that promote conjugation
Eg. Aggregation substance produced by pAD1 ( E. faecalis)catalyzes cell-cell contact
-potential recipients secrete small peptides that enter donor cells andstimulate transcription of pAD1 transfer genes
Microbial transfer of genes into eukaryotesEg. Agrobacterium tumefaciens- tumor inducing plasmid (Ti) that is
transferred to plant cells
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2. Transduction
DNA is transferred from cell to cell through a virus (bacteriophage)-amount of genes limited by what can fit in the phage head
Generalized transduction-take any gene from a donor cell and transfer it to a recipient cell(phage cannot distinguish between own DNA from host DNA)
Specialized transduction (temperate viruses)-can only transfer a few closely
linked genes between cells
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Generalized transduction (P1 phage)-virus must have a DNA packaging mechanism that allows accidental recognitionof host DNA
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Specialized transduction-linear phage-recircularizes at cohesive ends(cos sites)
-attP can combine with attB(located between gal and biogenes)-PROPHAGE
-reactivation of prophage
improper excision
-defective phageviral geneshost genes adjacent to the attB
phage
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Site-specific recombination
-requires very little sequence homology between the recombining DNAmolecules but does require a short (10-2bp) sequence recognized by the
recombination enzyme
(integrase)
f
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pSH2
fdh
M. smegmatis
cellHyg resistant
7H10 + Hyg
7H10 Hyg + Kan
pHINT
Hygrfdh
pSH1
Hyg & Kan resistant
Site specific mutagenesis
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Mutation: inherited change in the base sequence of the DNA comprising thegenome of the organism (mutant)
a. harmfulb. beneficialc. neutral
-Cell must fail to repair the change before replication
-genotype: the genetic makeup of an organismeg. hisC gene HisC proteinmutations is hisC: hisC1, hisC2
phenotype: the observable characteristics of an organism(could be different from parent strain)His+ His-
wildtype strain: strain isolated from nature
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Isolation of Mutants
-selectable mutations- confer an advantage to the organism; progeny
outgrows parent strain eg. Drug resistance-nonselectable mutations- may not result in a change in phenotype
Screening
Replica plating
Auxotroph
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Molecular basis of mutationA-spontaneousRates of mutations (DNA genomes)
errors in DNA replication- 10-7- 10-11/round of replication
transposition- 10-7
nonsense mutant- 10-6- 10-8
hot spots
Rates of mutations in RNA genomes-1000 fold higher - no RNA repair mechanisms
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P
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Point mutations-involving one or few base pairs (base pair substitution)Consequences?
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Transition-Purine to a different purine [ A to G ]-Pyrimidine to a different pyrimidine [ C to T]
Transversion-purines to pyrimidines or vice versa[C/ T A / G]
Missense mutationsLoss of function mutation
-decrease or eliminate activity of protein
Gain of function mutation-increase activity-expand substrate specificity
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Mutant 164 is mutated in mshC
164 CTCGACGCGTTG CCG TGGCGCGCCGAGCGTCC
WT CTCGACGCGTTG CTG TGGCGCGCCGAGCGTCC
nucleotide 554
ctg-leucine ccg-proline
5% ACTIVITY
n-METHYL n-NITROSOGUANIDINE
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Insertions and deletions-makes the sequence either longer or shorter-if the number of bases inserted or deleted is not a multiple of three,ribosome will read the wrong triplets
-premature stop codon-different protein sequence
Inversion-occurs when a fragment of DNA is
flipped in orientation relative to theDNA on either side
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Revertant-strain in which wild-type phenotype lost in the mutant is restored1. Same site revertant
2. Second site revertant-suppressor mutationa. a mutation somewhere else in the geneb. a mutation in another gene can restore the
enzyme functionc. a mutation in another gene can result in
another enzyme that results in a new metabolic pathway
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B.Inducedmutagenic treatment- increase rate of mutation
Mutation frequency= mutant cells present in a population
Mutagens-increase the mutation rate
-Chemical
-Physical
-Biological agents
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Mutagenesis1.Chemical mutagens-base analogs-substitutes look alike molecule
-alkylating agentseg. Nitrosoguanidine-introduce changes in nonreplicating DNA
-intercalating agents-insert between two
DNA base pairseg. Ethidium bromide
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2. Radiationa. nonionizing-UV (260 nm) absorbed by nucleotide bases resulting in production ofpyrimidine dimers (two adjacent pyrimidine bases become covalently joined)
b. ionizingshort wavelengths such as X-rays, cosmic rays and gamma rays-substances ionize-free radicals (hydroxyl radical) that react with DNA
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3.Biological and site-directed mutagenesis
a. Transposon mutagenesis
b. Mutations that arise from DNA repair-SOS regulatory system
-some repair occurs without a template-errors
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Transposable elements-transposition- the process by which a gene moves from one place to another in the genome (10-
5 - 10-7/ generation)1. transposase-recognizes, cuts, and ligates DNA2. short inverted terminal repeats at the end of the DNA
(20 bp- 1000bp)
A-insertion sequences (1000 bp)
- IS1, IS2.-found in plasmid, chromsomal, bacteriophage DNA
B-transposons- are insertion sequences with additional genes
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Target sequence is duplicated
-transposons can be random or prefer hot spots
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Ames Test
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Ames Test-bacteria as initial screen -hisG mutant-reversion mutation restores gene
-modify by treating potential mutagen with rat
liver extract
-inexpensive preliminary screen to weed outmutagenic chemicals
Transgenic mice
-mice with lacZ gene inserted into chromosomes-distributed throughout mouse-can find out if certain organs convert harmlessprecursor chemicals into mutagens
Salmonella - His- auxotroph
control
test
Disk with mutagen
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DNA Repair
-type of mutation-extent of damage
Error proof repair-rely on one of the two strands remainingundamaged
Eg. Base excision repair- snips damaged
bases from DNA
Eg. Recombinational repairone daughter strand is undamaged
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Error-Prone DNA repair-SOS response
-a coordinated cellular response to DNA damage
-in order to save the cell, can introduce mutations into severelydamaged DNA
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Gene regulation
How does the cell control all the different reactions involved in a
single cycle of cell growth?
How do microorganisms respond to changes in their environment?
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DNA sequence control-program the rearrangement of DNA so as to activate or disable a particulargene
Phase variation-DNA rearrangement to turn on and off expression of certain cell surfaceproteins
Fig. 9-1
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Transcription
No control
mRNAA
mRNAB
GeneA
Gene B
Translation
EnzymeA
Substrate
Product
Control of enzyme activity No product
Translational control No protein synthesis
Enzyme B
mRNA
C
Transcriptional control No mRNA synthesis
Gene C
Gene D
Fast
Slow
1 R l ti f ti it
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1. Regulation of enzyme activity
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Allosteric inhibition
1. Substrate binding site2. Allosteric site-inhibitor binding site
Covalent modificationEg. adenylation (the addition of AMP)
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Transcriptional control
Induction and repression1. Synthesis of enzymes involved in catabolic pathways can be inducible
-the initial substrate of the pathway (or some derivative of it) isusually the inducer
2. Synthesis of enzymes involved in anabolic pathways is repressible-end product of the pathway usually acts as a corepressor
Regulatory proteins- regulators bind specific small MW ligands to determine their concentrations- bind to specific DNA regulatory sequences
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1. Derepression of Lac Operon- catabolic
-genes that code for the enzymes needed for lactose catabolism
lactose glucose + galactose
Three Structural Genes-three enzymes in the lac catabolic pathway
LacY, LacZ, LacA,
Promoter- DNA segment where RNA polymerase binds and startstranscription
Operator- DNA segment found between the promoter and structuralgenes where the regulatory protein binds
Synthesis of the enzyme only when the substrate is present
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Synthesis of the enzyme only when the substrate is present
induction increases the amount of mRNA encoding the enzymes
Allolactose
-galactosidase
LacI
lacO
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Lac operon
-expressed at low level
-lactose transported into cell in low amounts-LacZ at low levels changes lactose into allolactose
-100X increase in expression of lac operon once lactose added to medium
2 Repression of arginine synthesis
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2. Repression of arginine synthesis(Anabolic pathway)
Total protein synthesis is
unchanged
repression decreases the amount of mRNA encoding the enzymes
Positive control
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-regulator protein promotes the binding of RNA polymerase andincreases mRNA synthesis
Eg.Maltose catabolism
Note: maltose catabolizing enzymes are spread out in various operons all
controlled by the activator protein-regulon
Activator sequence
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Two-Component Phosphorelay SystemsA signal transduction system that uses transfer of phosphoryl groups tocontrol gene transcription and protein activity
-sensor kinase protein- changes in conformation
- autophosphorylates at His residueeg. PhoQ in Salmonella senses magnesium
-response regulator- transphosphorylation at Glu orAsp residue
-phosphatase
Signal transduction and two component systems
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Environmental signal
Sensor kinase
Cytoplasmicmembrane
Response regulator
Transcription blockedDNA
Structural genesOperatorPromoter
RNApolymerase
Signal transduction and two component systems
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Global regulatory systems
-systems that affect many genes and pathways simultaneously, allowingfor both independent regulation of operons as well as cooperation ofoperons
a. a single regulator protein (repressor or activator) to regulate severaloperons
b. different sigma factors
c. nonprotein regulators-small regulatory mRNAs
Regulon-a collection of genes or operons controlled by a commonregulatory protein
R ul ti n b si m f ct rs
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Regulation by sigma factors
Bacteria produce a number of different sigma factors; each enables RNApolymerase to recognize and bind to specific promoters
Alteration of the sigma factors available to RNA polymerase changes geneexpression