Download What makes cells different from each other? How do cells respond to

  What makes cells different from each other?
  How do cells respond to information from environment?
  Regulation of:
- Transcription
- prokaryotes
- eukaryotes
- mRNA splicing
- mRNA localisation and translation
Diversity of cell types and functions
Red blood cells
Hair cells in cochlea
Cardiac muscle cells
Nerve
cells
Skin cells
What makes cells different is they make different proteins
Some proteins made only in specific cell types:
e.g., hemoglobin, insulin
Why do different cells make different proteins?
They all have the same genes…or do they?
How do we know that’s the case?
Can clone a new animal from many
different types of cells:
⇒  Each cell has all genes necessary to
make whole animal
(and all cell types within it)
Cloning => all cells
have same genes
All cells have same genes but not all genes “expressed”
in each cell
Housekeeping genes:
- expressed in all cells
(e.g., enzymes for cellular metabolism,
structural proteins)
Tissue-specific genes:
- expressed in just some cell types
(e.g., haemoglobin, insulin, liver enzymes,
neurotransmitter receptors, etc.)
Each cell type has a characteristic “profile” of expression
of different subsets of the 30,000 genes in the genome
How is production of proteins regulated?
  How is each protein regulated?
  How is the regulation of all proteins coordinated?
(differentiation)
  How do cells “know” which proteins to make?
(development)
  How is protein expression modulated?
(cellular metabolism / response to environment)
Various steps in protein expression can all be regulated:
- Transcription
(in which cells or under what conditions does
mRNA get made for gene X?)
- mRNA splicing
(many mRNAs must be spliced together from
separate “exons” to make mature message).
- mRNA localisation and translation
(where and when do you make protein X
from the mRNA?)
Core principles:
  Regulatory proteins bind control elements in DNA or RNA
  Control elements defined by specific sequences
  Both positive and negative regulation
  Mutations in either regulatory proteins or control elements
can cause defects in regulation
Fundamental principles of differential gene expression
first worked out in bacteria in the study of the response
to nutrient availability
Basic principles apply to all
organisms and to different
mechanisms of regulation
Differential gene regulation: the lac operon
Escherichia coli: preferred food source is glucose
Will grow on other sugars like lactose
Break it down to glucose and galactose
Induction of β-galactosidase
β-galactosidase protein normally hardly made at all
(in presence of glucose and absence of lactose)
But, in absence of glucose and presence of lactose:
10,000-fold induction in levels of β-galactosidase:
Growth on lactose depends on three enzymes:
β-galactosidase
Permease
Thiogalactoside transacetylase
(lacZ)
(lacA)
(lacY)
Each encoded by a different gene, but:
(i)  Genes are tightly linked on chromosome
(ii)  All are induced coordinately
(iii)  Ratio of Z:A:Y proteins remains constant
Conclude: under a common control system
(an “operon”)
Lac genes are co-transcribed in a single mRNA
promoter
Three proteins are translated separately
How does the induction occur?
How is the expression of these genes regulated in
response to environmental conditions?
How was this discovered?
Jacob and Monod (1961) isolated mutant strains of
E. coli with altered growth on lactose or altered
induction of lac genes:
Wild-type
gene
z+
y+
a+
Mutant
zya-
Growth on
lactose
-
Biochemical
phenotype
no galactosidase
no permease
no transacetylase
o+
oc
+
constitutive - all enzymes
always present
i+
iis
+
-
constitutive
repressed (non-inducible)
Dominance relationships and complementation
Used an episome F-lac to make a partial diploid
(a heterogenote)
Test whether mutation only has effect on genes
linked to it on the DNA:
- A mutation in a regulatory element should only affect
expression of genes right next to it (“in cis”)
-  A mutation in a gene encoding a regulatory protein
should affect expression of genes on another
chromosome (“in trans”)
F episome complementation
⇒  I makes some kind of diffusible substance
(LacI repressor protein)
⇒  The operator mutations only affect the expression
of genes with which they are linked
i.e., they only work in cis
⇒  O does NOT make a diffusible substance
Explanation of mutants
Mutant
zya-
Lactose
-
Explanation
no galactosidase
no permease
no transacetylase
oc
constitutive
Mutation in operator sequence repressor can not bind
i-
constitutive
Mutation in repressor it cannot bind operator
is
Is mutations
block binding
of lactose to
repressor
(always off)
-
Mutation in repressor - it cannot
bind lactose (always binds operator)
Lac operon also positively regulated
  Efficient binding of RNA polymerase to promoter
requires another protein:
- CAP (catabolite-activating protein)
  Binds to a specific sequence upstream of promoter
Catabolite repression (inhibiting the activator)
  Induction of lac operon allows cell to make glucose
from lactose
  But don’t need to do that when have glucose around
as well (don’t need to make more glucose)
  Activator shut off when glucose high
  CAP must be bound to cAMP to be active
  Glucose inhibits production of cAMP by adenylyl cyclase
  Known as “catabolite repression” because high levels
of catablolite (glucose) represses transcription
Lac operon also positively regulated
CAP…
O3
O1
O2
General principles from study of lac operon:
  Gene expression controlled by binding of proteins
to upstream regulatory elements (operators/enhancers)
  Each gene controlled by both activators and repressors
  Separate response elements for different proteins
  Mutations in regulatory proteins or in the DNA elements
can alter expression
  Expression regulated in response to external signals
(from environment or from other cells in embryo)
Lac operon is example of regulation of three linked genes
How do you coordinate regulation of larger sets of
(unlinked) genes?
Will examine this in fruitfly Drosophila melanogaster…