Post on 01-Jan-2016
transcript
Remember: Final Draft of Posters Due at 10 am on
Thursday!
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 Regulation is almost always based on initiation!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 Transcription
Some 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 together for regulation. But each has its own promoter and is individually its 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