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Anabaena sp. PCC 7120 je Nostoc
heterocysty nejsou cysty
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Paradox of developmental biology
How is it that a
single cell gives rise to a multicellular organism composed
of 100s of different cell types – yet all the cell types have the same genes?
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Patterning can involve the interpretation of positional information.
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Figure 21-63. Two strategies for using signal concentration
gradients to specify a fine-grained pattern of cells in
different states. In (A) there is only one signal gradient, and cells select their states by responding accurately to small changes of signal concentration. In (B) the initial signal gradient controls establishment of a small number of more local signals, which control establishment of other still more narrowly local signals, and so on. Because there are multiple local signals, the cells do not have to respond very precisely to any single signal in order to create the correct spatial array of cell states. Case B corresponds more closely to the strategy of the real embryo.
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Figure 21-65. The formation of ftz and eve
stripes in the Drosophila blastoderm. Genes ftz and eve
are both pair-rule genes. Their expression patterns (shown in brown for ftz and in gray for eve) are at first blurred but rapidly resolve into sharply defined stripes. (From P.A. Lawrence, The Making of a Fly. Oxford, UK: Blackwell, 1992.)
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The Course of Development
Time
Events in time and space
. . .
Complicated
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The Course of Development
Time
Complicated
Really
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Matveyev and Elhai (unpublished)
N2
Cyanobacteria
Anabaena grown without fixed nitrogen
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Paradox of developmental biology
How is it that a
single cell gives rise to a multicellular organism composed
of 100s of different cell types – yet all the cell types have the same genes?
How Cyanobacteria Count to 10
Robert Haselkorn
Jak každá desátá buňka ví že má být heterocystou
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N2 + 8H+ + 8e- + 16ATP -->
2NH3 + H2 + 16ADP + 16Pi
Note: Very expensive
Reason why N2 fixation by heterotrophic microbes is probably low
Key enzyme: nitrogenase (nif)
Ancient enzyme: highly conserved in very diverse microbes, from archaea to cyanobacteria
Biochemistry of N2 fixation
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What is another problem with nitrogenase?
Nitrogenase is killed
dead by O2
Protects nitrogenase (N2 fixing enzyme) from O2
Outside
sources of O2
O2 produced by cyanobacteria
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Ne všechny sinice schopné fixovat dusík tvoří heterocysty
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How does Trichodesmium (and single cell cyano’s) fix
N2 without heterocysts?
Partial answer: doesn’t fix N2 and do
photosynthesis at the same time
See Berman-Frank et al. Science (2001) 294: 1534-1537.
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1. Site of N2 fixation in many cyanobacteria.
2. Specialized thick wall cells in chain of cyanobacterial
vegetative cells
3. No PS II of photosynthesis --> no O2 evolution
4. No carbon fixation
5. Respiration
What are heterocysts?
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The heterocyst achieves a near anoxic state by
at least three means.
First, photosystem II, the O2-producing
end of the photosynthetic electron
transport chain, is dismantled during heterocyst differentiation, so that
the heterocyst need contend only against O2 produced by neighboring
vegetative cells and that dissolved in the environment. Second, heterocysts are
invested with a specialized envelope that limits the influx of gases.
Two layers within the envelope have been implicated in O2 protection:
an inner layer composed of a hydroxylated glycolipid and an outer layer
of polysaccharide. Neither layer is found in vegetative cells. Third, much of the O2
that overcomes these barriers is consumed by the high oxidase activity associated
with heterocysts.
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Excitation was at 510 to 560 nm (green),
exciting phycoerythrin, and emission was greater than 600 nm.
Heterocysts have negligible fluorescence, while vegetative cells have intense combined fluorescence from phycobiliproteins and chlorophyll a. Bar, 10 µm.
A médium s dusíkem
B médium bez dusíku
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Anabaena filament growing on nitrate
removal
of nitrate
18
hours
Heterocysts only
when needed
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Anabaena
heterocyst cells
vegetative cells
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Anabaena model
Heterocyst spacing relatively constant
Heterocyst cells
produce compound
Vegetative cells
divide
differentiate
consume
compound
diffuse compound
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First, they assumed that any cell is competent
to differentiate at the moment when nitrogen is removed
from the environment and that the choice of cells that initiate differentiation is random. Second, they postulated the existence of a diffusible inhibitor made by heterocysts and differentiating cells and consumed by nondifferentiating cells, as predicted by experimental data.
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Anabaena – continuous model
axiom: Fh(smax,cmax) Fv(smax,cmax) Fh(smax,cmax)
F(sl,cl)
Fv(s,c) > F(sr,cr):
if s < smax & c >
cmin
solve dc/dt = D.(cl+cr-2c)-µ.c
ds/dt = r . s
if s = smax & c > cmin
produce Fv(k . smax,c)Fv((1-k) . smax,c)
if c = cmin
produce Fh(s,c)
Fh(s,c):
solve ds/dt = rs . (smax-s)
dc/dt = rc . (cmax-c)
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Case of the Hidden Heterocyst
Matveyev and Elhai (unpublished)
N2
NH3
O2
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Case of the Hidden Heterocyst
Strategy to find heterocyst
differentiation genes
1. Use transposon mutagenesis
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Case of the Hidden Heterocyst
Strategy to find heterocyst
differentiation genes
Nostoc genome
Transposon
1. Use transposon mutagenesis
to find a mutant
defective in heterocyst differentiation
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Case of the Hidden Heterocyst
Strategy to find heterocyst
differentiation genes
Nostoc genome
2. Sequence out from transposon
AAGCTTGACCAAAAAGTTAAAACACTGACGGCAAATAATCAATGACTATCAGACAGAGAATCATCGTGCTGTCAGTAAAACCTCTGATTTCGATCTTTACCATAATTGTTATGTTGTAATGACTAACCAGACTATCTTTTACAGAGCTTCTGGTTAACACTTGTCTAATTAGACATTGATAATGTTTGTGGGGGTTGGTCATCAGGAATGGTAAATAGCAATTACCCTTCAGACTTTCCTATGAGACGCTCCGCCAACGAGCAGTGTCTCTTAAAGAACGTTATGAGCGCTCAGTTAACTTCAGAAATTCACGGCGGAAATCCATAGTTATTATTACTTATGACTAAAACAAAATTACTATGGCGGCTTGTTTAATATAGATTCTGTGTTCTGAGAAATGACTTTTAAAGTCCCACTAACTTTTTTCTCATCTATTGCTATATTTCGACTTTAAAACTTATAGTAGATGGCTTAATTCTCAAATAACAAACTCATTTTTAGTAGATATTTCATGCAAACTGAGGTTTTTAGTGATATTTTCCCCTTATTGAGTACAGCCACTCCACAAACCTTAGAATGGCTACTCAATATTGCAATTGATCATGAATATCCCACTGGTAGAGCAGTTTTAATGGAAGATGCCTGGGGTAATGCAGTTTATTTCGTTGTATCTGGATGGGTAAAAGTTCGGCGCACCTGTGGA
1. Use transposon
mutagenesis
to find a mutant defective in heterocyst differentiation
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Case of the Hidden Heterocyst
Strategy to find heterocyst
differentiation genes
Nostoc genome
2. Sequence out from transposon
AAGCTTGACCAAAAAGTTAAAACACTGACGGCAAATAATCAATGACTATCAGACAGAGAATCATCGTGCTGTCAGTAAAACCTCTGATTTCGATCTTTACCATAATTGTTATGTTGTAATGACTAACCAGACTATCTTTTACAGAGCTTCTGGTTAACACTTGTCTAATTAGACATTGATAATGTTTGTGGGGGTTGGTCATCAGGAATGGTAAATAGCAATTACCCTTCAGACTTTCCTATGAGACGCTCCGCCAACGAGCAGTGTCTCTTAAAGAACGTTATGAGCGCTCAGTTAACTTCAGAAATTCACGGCGGAAATCCATAGTTATTATTACTTATGACTAAAACAAAATTACTATGGCGGCTTGTTTAATATAGATTCTGTGTTCTGAGAAATGACTTTTAAAGTCCCACTAACTTTTTTCTCATCTATTGCTATATTTCGACTTTAAAACTTATAGTAGATGGCTTAATTCTCAAATAACAAACTCATTTTTAGTAGATATTTCATGCAAACTGAGGTTTTTAGTGATATTTTCCCCTTATTGAGTACAGCCACTCCACAAACCTTAGAATGGCTACTCAATATTGCAATTGATCATGAATATCCCACTGGTAGAGCAGTTTTAATGGAAGATGCCTGGGGTAATGCAGTTTATTTCGTTGTATCTGGATGGGTAAAAGTTCGGCGCACCTGTGGA
1. Use transposon
mutagenesis
to find a mutant defective in heterocyst differentiation
3. Find gene boundaries
4. Identify gene
Do it
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HetR
mutant - unable to make heterocysts
The spatially patterned
differentiation of heterocysts in the filamentous cyanobacterium Anabaena requires
a functional hetR gene
low level of transcript when Anabaena is grown with combined nitrogen
induction begins within 2 h following nitrogen deprivation
by 3.5 h, induction is localized to spaced foci
by 6 h, 20-fold increase within spatially separated cells
positive autoregulation
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HetR
Genes
needed for differentiation
Master
regulator
Differentiation in cyanobacteria
Integration of
signals through HetR
Position in filament
Position in cell cycle
Nitrogen deprivation
????
??
NNNNNNNNNNNNNNNNNNATGNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNTACNNNNNNNNNNNNNNNN
hetR gene
How might hetR be controlled?
5’-GTANNNTACNNNNNNNNNNTANNNTNNNNNNNNNN
3’-CATNNNATGNNNNNNNNNNATNNNANNNNNNNNNN
Presence of fixed nitrogen
No HetR protein
Transcription
Absence of fixed nitrogen
NNNNNNNNNNNNNNNNNNATGNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNTACNNNNNNNNNNNNNNNN
hetR gene
5’-GTANNNTACNNNNNNNNNNTANNNTNNNNNNNNNN
3’-CATNNNATGNNNNNNNNNNATNNNANNNNNNNNNN
RNA Polymerase
Absence of fixed nitrogen
How might hetR be controlled?
NNNNNNNNNNNNNNNNNNATGNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNTACNNNNNNNNNNNNNNNN
hetR gene
5’-GTANNNTACNNNNNNNNNNTANNNTNNNNNNNNNN
3’-CATNNNATGNNNNNNNNNNATNNNANNNNNNNNNN
RNA Polymerase
Absence of fixed nitrogen
How might hetR be controlled?
HetR protein
Transcription
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The key result of this experiment is that
all of the upstream controls of HetR expression can
be bypassed; expression of HetR alone suffices to turn on the differentiation pathway.
HetR overexpression
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PatS
overexpression of patS completely blocks heterocyst development
patS encode
a 17- or 13-amino-acid peptide, is crucial for the
formation and maintenance of the normal heterocyst pattern
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Wild-type filaments (A) grown in BG-11 medium and
(B) after the nitrogen step-down in BG-110 to induce
heterocysts (arrowheads) are shown. (C) Overexpression of patS prevented heterocyst formation in BG-110, and (D) deletion of patS resulted in supernumerary heterocysts with an abnormal pattern in BG-110. Differential interference contrast micrographs were taken before (A) and 24 hours after (B through D) heterocyst induction.
patS controls heterocyst development in Anabaena PCC 7120
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The exogenous addition of a pentapeptide corresponding to
the last five COOH-terminal residues of PatS also inhibited
heterocyst differentiation, indicating that a processed form of PatS may be a diffusible inhibitory signal regulating development.
R G S G R
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Přesně jako v modelu
z roku 1975
The inhibition of neighboring cells by select differentiating cells (lateral inhibition) is an important mechanism of pattern formation in eukaryotic organisms.
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Because it takes ~20 hours for heterocysts to
mature and begin supplying fixed nitrogen to the filament,
a specialized early inhibitory signal is required to allow only a fraction of starving cells to terminally differentiate.
The first cells to differentiate increase the production of PatS to inhibit neighboring cells from forming heterocysts. PatS-producing cells must themselves be refractory to the PatS signal.
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+ Nostoc punctiforme
Anthoceros punctatus.
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Další události nezbytné k aktivaci nif genů