2 component regulatory systems
•
Maltose=effector, BUT if signal not DIRECTLY involved, but needs
to be transmitted and changed = signal transduction
•
Sensor protein=
–
kinase, phosphorylates compounds,
–
membrane associated
•
Phosphoryl group transmitted to another regulator IN the cell
–
Often a DNA binding protein involved in transcription
•
Many examples, N
-
fixation, sporulation,chemotaxis
Chemotaxis
•
Attractants decrease rate of autophosphorylation
•
Repellant increased autophosphorylation
•
CheA
-
CheW=transducer
•
CheY controls switch
–
cheY
-
P tumbles, CCW
-
CW
•
CheB phosporylated by CheA
-
P, but slower response than
CheY
-
P
•
CheB involved methylation
–
Fully methylated = best for repellants
–
cheB
-
P demethylates, occurs when attractants High
–
Degree of methylation regulates attraction/repulsion
Chemotaxis
Genetic exchange: transformation
Genetic exchange: plasmid transfer
•
Small, usually circular, independently replicating DNA
molecules
–
Generally, G
-
plasmids replicate as does chromosomal DNA, G
+
plasmids by “rolling circle” replication
•
Genes of replication control, timing initiation on plasmid (
ori
)
–
Some plasmids integrate (F
+
, Hfr)
–
Most are double
-
stranded
–
About 1
-
100kb
•
Code for:
–
R
-
factors (R
-
plasmids) : antibiotic resistance, heavy metal
resistance
–
Virulence plasmids : adhesins, hemolytic factors, toxin, Ti,
bacteriocins
–
Degradation, tol, nah,
•
Plasmid copy #
•
Compatibility (
inc
)
Genetic exchange: plasmid transfer
•
Small, usually circular, independently replicating DNA
molecules
–
Generally, G
-
plasmids replicate as does chromosomal DNA, G
+
plasmids by “rolling circle” replication
•
Genes of replication control, timing initiation on plasmid (
ori
)
–
Some plasmids integrate (F
+
, Hfr)
–
Most are double
-
stranded
–
About 1
-
100kb
•
Code for:
–
R
-
factors (R
-
plasmids) : antibiotic resistance, heavy metal
resistance
–
Virulence plasmids : adhesins, hemolytic factors, toxin, Ti,
bacteriocins
–
Degradation, tol, nah,
•
Plasmid copy #
•
Compatibility (
inc
)
Avery Experiment
Transformation
•
Competence
: ability to be transformed
•
Steps in transformation
–
DNA binding and uptake (SS or DS, depending
on species)
–
Integration (recA)
•
Competence may be induced by
electroporation, Ca
Mechanism of transformation
Transduction
•
Generalized
—
low frequency
•
Specialized
—
high frequency:requires
specific integration
Generalized transduction
Specialized transduction
Specialized transduction
Genetic exchange: conjugation
Conjugation: early
Cell
-
surface
structure
Conjugation: middle
Conjugation: late
Genetic Engineering
-
basics
•
Basic steps in cloning
–
Restriction
-
modification enzymes
–
“shot gun” or PCR
–
Ligase
–
Recombination Plasmids (or phage)=
vectors
–
Expression vectors
•
Selection of clones
•
Looking for a clone with a specific gene
–
Probes (DNA, RNA or antibody)
•
Wave of the future: DNA chips or “microarrays”, BAC
libraries, automated sequencing etc
Genomics
•
Bioinformatics
•
Harvesting genes for biotech (Diversa)
–
Recent Science article
•
Comparing gene families
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