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Today:•Poster Hints•Microbial Genetics•Gene Regulation•Group Quiz 8

An Introduction to Microbial Genetics

Bacteria Reproduce Asexually via

BINARY FISSION

Can this generate genetic

diversity?!?

But, Bacteria still undergo GENETIC RECOMBINATION (combining DNA from two

individuals into the genome of a single individual)

Genetic Recombination in bacteria occurs through three distinct processes:

1. TRANSFORMATION

2. TRANSDUCTION

3. CONJUGATION

1.TRANSFORMATION-Def: The uptake of naked, foreign DNA

from the surrounding environment.• The foreign DNA may be incorporated into

the bacterial chromosome.• Some bacterial cells have specialized surface

proteins for the uptake of DNA.

General notes about PLASMIDS…

•Plasmids are small, circular, self-replicating DNA molecules separate from the bacterial chromosomes.•Some plasmids, like the F plasmids, can reversibly incorporate into the cell’s chromosome (an episome).

2. TRANSDUCTION

•Generalized Transduction occurs when the phage accidentally transfers random

bacterial genes instead of its own.•Specialized Transduction occurs when the

phage takes a small adjacent region of the bacterial DNA with it.

Def: Phage (bacterial viruses)

carry bacterial genes from one

host cell to another.

3. CONJUGATION

•Transfer is one-way, with the DNA donor (or “male”) attaching to the DNA recipient (“female”) with a sex pilus.•The ability to form sex pili and donate DNA results from the presence of an F factor.

Def: The direct transfer of genetic material between two bacterial cells

that are temporarily joined.

R PLASMIDSR plasmids contain genes conferring

resistance to antibiotics.

R plasmids, like F plasmids, have

genes that encode sex pili

and enable transfer from one cell to

another.Many R plasmids carry multiple

antibiotic resistance genes.

R Plasmids acquire multiple resistance genes through TRANSPOSONs.

Transposon: a piece of DNA that can move from one location to another in a cell’s genome.

Transposase catalyzes the movement of the transposon from one location to another

Composite Transposons include extra genes sandwiched between two insertion sequences.

Composite transposons can add a gene conferring antibiotic resistance to a plasmid already carrying genes for resistance. In an environment high in antibiotics, natural selection will favor multi-drug

resistant bacterial clones.

Gene Regulation:

Individual bacteria cope

with environmental fluctuation at several

levels:

Regulation of Gene Expression occurs through OPERONS.

OPERONS utilize a segment of DNA called an OPERATOR. The operator controls the access

of RNA polymerase to the genes.

OPERATORS function as switches to turn transcription ON or OFF.

Operators are ON unless a specific REPRESSOR PROTEIN is bound to it.

This operon, the trp operon is an example of a REPRESSIBLE OPERON because

transcription is INHIBITED when tryptophan binds to the regulatory protein.

INDUCIBLE OPERONs are STIMULATED when a specific small molecule interacts

with a regulatory protein.Examples of Inducible Operons:

•the lac operon

•the ara operon

INDUCIBLE OPERONS: The lac Operon

The lac OPERON is also positively regulated by cyclic AMP (cAMP) and the regulatory protein, cAMP receptor protein (CRP).

Activated CRP stimulates transcription.

Thinking About Genomes…Thinking About Genomes…

Understanding Understanding Genome Genome

Structure and Structure and Function! Function!

Why is genome structure/ function

important?

Remembering Structure…Remembering Structure…

Nucleosomes are formed of DNA winding Nucleosomes are formed of DNA winding around 8 histone proteins, two each of H2A, around 8 histone proteins, two each of H2A, H2B, H3, and H4. The N-terminus of each H2B, H3, and H4. The N-terminus of each

protein extends outward forming a “histone protein extends outward forming a “histone tail”.tail”.

Nucleosomes condense into 30nm fibers Nucleosomes condense into 30nm fibers due to interactions between the histone due to interactions between the histone tails of one nucleosome, the linker DNA, tails of one nucleosome, the linker DNA,

and the nucleosomes on either side.and the nucleosomes on either side.

Remembering Structure…Remembering Structure…

During prophase, chromosomes may condense further!During prophase, chromosomes may condense further!

Remembering Structure…Remembering Structure…

Thinking About Genomes…Thinking About Genomes…In metaphase In metaphase chromosomes, chromosomes,

the same genes the same genes always end up at always end up at

the same the same locations. What locations. What does this tell us does this tell us

about about chromosome chromosome

packing??packing??Photo: V. Miszalok, U. Klingbeil, I. Chudoba, V.

Smolej

The Importance of The Importance of Gene ExpressionGene Expression

Cell Differentiation! Cell Differentiation! Differences in cell types are Differences in cell types are

due to differential gene due to differential gene expression.expression.

How might a cell regulate

gene expression??

Gene Regulation at the Level Gene Regulation at the Level of Chromatin Structureof Chromatin Structure

Heterochromatin vs Euchromatin??Heterochromatin vs Euchromatin??

Regulating Regulating ChromatinChromatin

Histone Histone acetylation (-acetylation (-

COCHCOCH33) ) prevents prevents adjacent adjacent

nucleosomes nucleosomes from binding to from binding to

one anotherone anotherSo does this activate or silence the acetylated

region?

Regulating ChromatinRegulating Chromatin

Staining of Acetylated H3 Throughout the Cell Cycle. A field of cells containing interphase, prophase(P), prometaphase (PM) and metaphase (M); Michael J. Hendzel and Michael J. Kruhlak

Other Modifications to Histone TailsOther Modifications to Histone Tails Histones may also be Histones may also be

Methylated (CHMethylated (CH33)) New Model: Histone New Model: Histone

Code Hypothesis!Code Hypothesis!

Figure: two different metaphase spreads (human female) with preferential staining.  Barbara A. Boggs, Peter Cheung, Edith Heard, David L. Spector, A. Craig Chinault & C. David Allis

Remembering DNA MethylationRemembering DNA Methylation

Proteins that bind Proteins that bind to methylated to methylated

DNA may recruit DNA may recruit histone histone

deacetylasesdeacetylases!!

What would this enzyme do??

New Field: Epigenomics!New Field: Epigenomics!

Figure: Epigenomics.com

Next Up: Next Up: Regulating at the Regulating at the

Level of Level of Transcription!Transcription!

Regulating TranscriptionRegulating Transcription

Regulation is almost always based on initiation!Regulation is almost always based on initiation!

Regulating Transcription: Regulating Transcription: Transcription FactorsTranscription Factors

Transcription factors Transcription factors may be may be generalgeneral (required for (required for transcription of all transcription of all genes) or genes) or specificspecific, required , required for high level for high level expression of expression of particular genes)particular genes)

Regulating Regulating Transcription: Transcription:

Enhancers Enhancers

and and ActivatorsActivators

Evidence for EnhancersEvidence for EnhancersMichael R. Botchan and his Michael R. Botchan and his

colleagues have produced colleagues have produced visual evidence of this model visual evidence of this model of enhancer action. They of enhancer action. They created an artificial DNA created an artificial DNA molecule and observed how molecule and observed how it interacted with enhancers it interacted with enhancers using an electron microscopeusing an electron microscope

Regulating TranscriptionRegulating TranscriptionSome Some specific specific transcription transcription

factorsfactors function function as as repressorsrepressors!!

Common Mechanisms:• Block binding of activators• Bind to their own control elements• Recruit histone deacetylases

Combinatorial Combinatorial ControlControl

Numbers and diversity Numbers and diversity of control elements of control elements

suggest that a suggest that a particular particular

combination of combination of control elements control elements

regulates regulates transcription.transcription.

Gene Clusters for Coordinated Gene Clusters for Coordinated ControlControl

Eukaryotic genes may also be packaged Eukaryotic genes may also be packaged together for regulation. But each has its together for regulation. But each has its own promoter and is individually own promoter and is individually transcribed.transcribed.

More often, genes in a pathway are More often, genes in a pathway are scatted on multiple chromosomes, but scatted on multiple chromosomes, but respond to the same combination of respond to the same combination of control elements.control elements.

Post-Post-Transcriptional Transcriptional

RegulationRegulation

Post-Transcription: RNA Post-Transcription: RNA ProcessingProcessing

(This helps explain our embarrassingly (This helps explain our embarrassingly small number of genes!)small number of genes!)

Post-Post-Transcription: Transcription:

mRNA mRNA DegradationDegradation

Shortening the Shortening the poly-A tail can poly-A tail can trigger trigger removal of the removal of the 5’ cap, 5’ cap, followed by followed by breakdown of breakdown of the mRNAthe mRNA Figure: Effect of TTP deficiency on the

stability of TNF-a mRNA in bone-marrow derived macrophages J. Blackshear, Biochem. Soc. Trans.. (2001) 30, (945–952)

Post-Transcription: RNA Post-Transcription: RNA Interference (RNAi)Interference (RNAi)

Experimental Observation: Injecting Experimental Observation: Injecting dsRNA into a cell can silence the dsRNA into a cell can silence the

corresponding gene!corresponding gene!http://www.nature.com/focus/rnai/animations/animation/animation.htm

Post-Post-Transcription: Transcription:

Initiation of Initiation of TranslationTranslation Regulatory proteins can block Regulatory proteins can block

attachment of ribosomesattachment of ribosomes Lack of poly-A tails blocks translationLack of poly-A tails blocks translation Block may be global (all translation; Block may be global (all translation;

i.e. egg cells, dormant plants)i.e. egg cells, dormant plants)

Post-Transcription: Protein Post-Transcription: Protein Processing and DegradationProcessing and Degradation

Many proteins must be cleaved or modified Many proteins must be cleaved or modified to become active. to become active.

Proteins may also be labeled for destruction Proteins may also be labeled for destruction by attaching a small protein, by attaching a small protein, ubiquitinubiquitin, , that attracts that attracts proteosomesproteosomes

Genome Regulation and CancerGenome Regulation and Cancer Viruses may carry Viruses may carry oncogenesoncogenes (genes (genes

causing cancer) causing cancer) Oncogenes are similar to Oncogenes are similar to proto-oncogenesproto-oncogenes

(genes involved in normal cell division) in our (genes involved in normal cell division) in our genomegenome

A Multistep Model for A Multistep Model for CancerCancer