2 component regulatory systems

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