Chapter 8: Microbial Genetics

Genome: genetic information in a cell

Chromosome: contains the genes-segments of DNA

DNA: nucleotide base pairs - genetic code for proteins

Central Dogma:DNA - RNA - ProteinsGenomics: sequencing

and molecular characterization of genomes

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Bacteria

Single circular chromosome

Looped and folded and attached at one or several points to the plasma

membrane

THAT SPELLS DNA

Jonathan Coulton

http://www.jonathancoulton.com/store/downloads/

We start the story when Mom met DadAnd they danced all night and he took her homeIt might have been all the wine they hadBut they rolled the dice and won your genome

Then you grew and you grew and one day you were youAnd you looked like your father and motherIF you’re looking for someone convenient to blameYou can take your pick it’s one or the other

DNA, you’re in my heartDNA, in fact you’re in every part of my bodyEach cell has a nucleus, each nucleus has chromosomesAnd DNA, baby, that spells DNA

Guanine met Cytosine, fell in loveAnd then Thymine got busy with AdenineThey sent the messenger-RNATo the ribosome to make more protein

And while it’s killing you dead it will mess with your headAnd it’s the light in the dark that will guide youIt’s the pages and pages of what you are like In the giant book that’s hidden inside you

DNA, you’re in my heartDNA, in fact you’re in every part of my bodyEach cell has a nucleus, each nucleus has chromosomesAnd DNA, baby, that spells DNA

If it says TGGTCGAACThen you might get the cancerIf it says GTCACGACAGGThen you shouldn’t eat shrimp or nutsIf it says TATACACATATCCTCGTThen you’ll probably wish that you didn’t know

The time will come when you’re almost goneAnd you try to guess but you’ll never knowYou do your best and you soldier onEvery day you’re here till it’s time to go

All the good things and bad that you do or don’t haveYou can find out for sure if you got’emBut there’s a spiraling staircase that you’re falling downAnd you’re nothing but dead at the bottom

DNA, you’re in my heart

DNA, in fact you’re in every part of my body

Each cell has a nucleus, each nucleus has chromosomes

And DNA, baby, that spells DNA

DNA RNA PROTEINS Replication Transcription Translation

DNA RNA PROTEINS • Semiconservative replication: each new DNA

molecule contains one original and one new strand

• Requires TONS of energy - supplied by the nucleotides (nucleoside triphosphates)

*Two phosphates are removed to add the nucleotide to a

growing stand

*Hydrolysis (exergonic)

• Accurate: mistakes made in 1 in every 1010 bases ***Proofreading-DNA pol

Replication Transcription Translation

DNA RNA PROTEINS Bacteria: • Some replicate bidirectionally around the chromosome• Two replication forks move in opposite direction away

from the origin of replication• Eventually meet when replication is completeEx: E.coli

Replication Transcription Translation

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TRANSCRIPTION

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GENE REGULATION

• Turning on and off genes• Save energy and resources• Repression: Turns OFF

– Response to overabundance of an end product– Repressors block RNA pol

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GENE REGULATION

• Induction: Turns ON– Inducer: acts to induce transcription– EX: lac operon

• E. coli (bacteria that live in intestines of mammals)• Discovered how genes control the metabolism of the sugar

lactose• Lactose: disaccharide made of glucose and galactose• Milk sugar

– http://www.youtube.com/watch?v=oBwtxdI1zvk&feature=related

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When there is/is not lactose

LACTOSE PRESENT: It induces E. coli to produce three enzymes which will metabolize lactose

LACTOSE ABSENT: three enzymes will not be made. Lactose will not be broken down.

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DNA of E.coli

Regulator gene: codes for repressor genePromoter: DNA segment that is recognized by the enzyme RNA

polOperator: DNA segment that serves as a “switch” controlling the

access of RNA pol to the promoterStructural genes: code for 3 enzymes that break down lactose

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Operon “turned off”• Repressor protein attaches to the operator

physically blocking RNA pol from attaching

RNA pol

Operon “turned on”

• Lactose present (acts as inducer starts gene expression)

• Binds to the repressor protein

• Repressor changes shapes and detaches from operator

• RNA pol is not blocked anymore

• All three enzymes can be produced

Operator “turned on”

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lactose

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• E. coli is able to turn off and on genes when lactose is present/absent

• Bacteria saves resources and produce only those proteins that are needed

Let’s watch it again

• http://www.youtube.com/watch?v=oBwtxdI1zvk&feature=related

MuTaTiOnS

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• GOOD– New enhanced activity

that benefits the cell

• BAD– Inactive or less active,

lethal?

• SILENT(neutral)– No change in activity

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Types of Mutations

Base Substitution (point)• Single base in DNA is replacedMissense mutation: substitution results in an

amino acid substitutionNonsense mutation: creates a stop codon in the

middle of mRNA....preventing synthesis of protein

Types of Mutations

Frameshift• One or a few nucleotide pairs are deleted or inserted

in the DNA• Change the reading frame of codons

*Mutations can occur spontaneously or by mutagens

MUTAGENS• Chemical:

– Nitrous acid (HNO2): causes A to bond with C instead of T

MUTAGENS

• Radiation: x-rays, UV rays, gamma rays– Cause electrons to pop out of their usual shells– Ions can combine with bases in DNA, resulting in errors in

DNA– Breakage of covalent bonds in sugar-phosphate backbone-

breaks in chromosomes

• Exposure to UV causes T’s to cross link forming a thymine dimer

• Unless repaired, these may cause serious damage or death to the cells because it cannot properly transcibe or replicate.

• Some organisms have enzymes that can repair these dimers

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Genetic Transfer and Recombination1. Conjugation: transfer of a plasmid

2. Transduction: transfer of DNA via a virus

3. Transformation: genes transferred from one bacterium to another as “naked” DNA

Conjugation

Movement of a plasmid

b/w two cellsPlasmid: small, self-replicating,

gene-containing circular piece of DNA

1. Requires direct cell to cell contact

2. Opposite mating types (donor cell has a plasmid, recipient cells do not)

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Transduction

• Bacterial DNA is transferred from a donor cell to recipient inside a virus that infects bacteria

• Bacteriophage (phage)

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•First done 70 yrs ago: Griffith•http://www.dnatube.com/video/997/Transformation

HOW DOES THIS HAPPEN?

Can happen naturally after cell death and cell lysis• Cell wall becomes permeable to large DNA molecules• Use surface proteins and type 4 pili to extend and take it in• Works best when donor and recipient cells are closely related

Artificial transformation:– Competence: when recipient cell is in a physiological state

to take up donor DNA– Changing the ionic strength of the medium and heating the

cells in the presence of positive ions (Calcium)– Makes cell membrane permeable

We are going to do this!!!

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pBLU Transformation

• Use an ampicillin-sensitive strain of E. coli, incapable of producing b-galactosidase for lactose breakdown

• Induce it to take up pBLU plasmid DNA

• Plasmid contains genes for Ampicillin resistance and the entire b-glactosidase gene

• http://www.biologie.uni-hamburg.de/b-online/library/onlinebio/14_1.jpg