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Презентация на тему Diferenciace heterocyst

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Anabaena sp. PCC 7120 je Nostocheterocysty nejsou cysty
Diferenciace heterocyst  Martin Tichý Martin Tichý Institute of Microbiology, Czech Anabaena sp. PCC 7120 je Nostocheterocysty nejsou cysty Paradox of developmental biologyHow is it that a single cell gives rise Patterning can involve the interpretation of positional information. Figure 21-63. Two strategies for using signal concentration gradients to specify a Expression of eve Stripe 2 Figure 21-65. The formation of ftz and eve stripes in the Drosophila The Course of Development The Course of DevelopmentTimeEvents in time and space . . .Complicated The Course of DevelopmentTimeComplicatedReally Matveyev and Elhai (unpublished)N2CyanobacteriaAnabaena grown without fixed nitrogen Paradox of developmental biologyHow is it that a single cell gives rise Fixace dusíku N2 + 8H+ + 8e- + 16ATP -->		 2NH3 + H2 + What is another problem with nitrogenase?Nitrogenase is killed dead by O2Protects nitrogenase Ne všechny sinice schopné fixovat dusík tvoří heterocysty How does Trichodesmium (and single cell cyano’s) fix N2 without heterocysts?Partial answer: 1. Site of N2 fixation in many cyanobacteria. 2. Specialized thick wall The heterocyst achieves a near anoxic state by at least three means. Dělba práce Excitation was at 510 to 560 nm (green), exciting phycoerythrin, and emission Anabaena filament growing on nitrateremoval  of nitrate18 hoursHeterocysts only when needed Anabaenaheterocyst cellsvegetative cells Anabaena modelHeterocyst spacing relatively constantHeterocyst cellsproduce compoundVegetative cellsdividedifferentiateconsume compounddiffuse compound First, they assumed that any cell is competent to differentiate at the Anabaena – continuous modelaxiom: Fh(smax,cmax) Fv(smax,cmax) Fh(smax,cmax)F(sl,cl) < Fv(s,c) > F(sr,cr):	if s Anabaena – continuous model Case of the Hidden HeterocystMatveyev and Elhai (unpublished)N2NH3O2 Case of the Hidden HeterocystStrategy to find heterocyst differentiation genes1. Use transposon mutagenesis Case of the Hidden HeterocystStrategy to find heterocyst differentiation genesNostoc genomeTransposon1. Use Case of the Hidden HeterocystStrategy to find heterocyst differentiation genesNostoc genome2. Sequence Case of the Hidden HeterocystStrategy to find heterocyst differentiation genesNostoc genome2. Sequence HetRmutant - unable to make heterocystsThe spatially patterned differentiation of heterocysts in HetRGenes  needed for differentiationMaster  regulatorDifferentiation in cyanobacteria Integration of signals Promoter The key result of this experiment is that all of the upstream PatSoverexpression of patS completely blocks heterocyst developmentpatS encode a 17- or 13-amino-acid Wild-type filaments (A) grown in BG-11 medium and (B) after the nitrogen The exogenous addition of a pentapeptide corresponding to the last five COOH-terminal Přesně jako v modeluz roku 1975 Because it takes ~20 hours for heterocysts to mature and begin supplying + Nostoc punctiformeAnthoceros punctatus. Další události nezbytné k aktivaci nif genů
Слайды презентации

Слайд 2 Anabaena sp. PCC 7120 je Nostoc


heterocysty nejsou cysty

Anabaena sp. PCC 7120 je Nostocheterocysty nejsou cysty

Слайд 4 Paradox of developmental biology

How is it that a

Paradox of developmental biologyHow is it that a single cell gives

single cell gives rise to a multicellular organism composed

of 100s of different cell types – yet all the cell types have the same genes?

Слайд 5 Patterning can involve the interpretation of positional information.

Patterning can involve the interpretation of positional information.

Слайд 6 Figure 21-63. Two strategies for using signal concentration

Figure 21-63. Two strategies for using signal concentration gradients to specify

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.

Слайд 7 Expression of eve Stripe 2

Expression of eve Stripe 2

Слайд 8 Figure 21-65. The formation of ftz and eve

Figure 21-65. The formation of ftz and eve stripes in the

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.)

Слайд 9 The Course of Development

The Course of Development

Слайд 10 The Course of Development
Time
Events in time and space

The Course of DevelopmentTimeEvents in time and space . . .Complicated

. . .
Complicated


Слайд 11 The Course of Development
Time
Complicated
Really

The Course of DevelopmentTimeComplicatedReally

Слайд 12 Matveyev and Elhai (unpublished)
N2

Cyanobacteria
Anabaena grown without fixed nitrogen

Matveyev and Elhai (unpublished)N2CyanobacteriaAnabaena grown without fixed nitrogen

Слайд 13 Paradox of developmental biology

How is it that a

Paradox of developmental biologyHow is it that a single cell gives

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


Слайд 14 Fixace dusíku

Fixace dusíku

Слайд 15

N2 + 8H+ + 8e- + 16ATP -->

N2 + 8H+ + 8e- + 16ATP -->		 2NH3 + H2

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


Слайд 16 What is another problem with nitrogenase?
Nitrogenase is killed

What is another problem with nitrogenase?Nitrogenase is killed dead by O2Protects

dead by O2
Protects nitrogenase (N2 fixing enzyme) from O2
Outside

sources of O2
O2 produced by cyanobacteria

Слайд 17
Ne všechny sinice schopné fixovat dusík tvoří heterocysty

Ne všechny sinice schopné fixovat dusík tvoří heterocysty

Слайд 21 How does Trichodesmium (and single cell cyano’s) fix

How does Trichodesmium (and single cell cyano’s) fix N2 without heterocysts?Partial

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.

Слайд 22 1. Site of N2 fixation in many cyanobacteria.

1. Site of N2 fixation in many cyanobacteria. 2. Specialized thick


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?


Слайд 23 The heterocyst achieves a near anoxic state by

The heterocyst achieves a near anoxic state by at least three

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.

Слайд 24 Dělba práce

Dělba práce

Слайд 25 Excitation was at 510 to 560 nm (green),

Excitation was at 510 to 560 nm (green), exciting phycoerythrin, and

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


Слайд 26 Anabaena filament growing on nitrate
removal of nitrate
18 hours
Heterocysts only

Anabaena filament growing on nitrateremoval of nitrate18 hoursHeterocysts only when needed

when needed


Слайд 27 Anabaena
heterocyst cells
vegetative cells

Anabaenaheterocyst cellsvegetative cells

Слайд 29 Anabaena model
Heterocyst spacing relatively constant
Heterocyst cells
produce compound
Vegetative cells
divide
differentiate
consume

Anabaena modelHeterocyst spacing relatively constantHeterocyst cellsproduce compoundVegetative cellsdividedifferentiateconsume compounddiffuse compound

compound
diffuse compound


Слайд 30 First, they assumed that any cell is competent

First, they assumed that any cell is competent to differentiate at

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.

Слайд 31 Anabaena – continuous model
axiom: Fh(smax,cmax) Fv(smax,cmax) Fh(smax,cmax)
F(sl,cl)

Anabaena – continuous modelaxiom: Fh(smax,cmax) Fv(smax,cmax) Fh(smax,cmax)F(sl,cl) < Fv(s,c) > F(sr,cr):	if

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)




Слайд 32 Anabaena – continuous model

Anabaena – continuous model

Слайд 33 Case of the Hidden Heterocyst
Matveyev and Elhai (unpublished)
N2
NH3

O2

Case of the Hidden HeterocystMatveyev and Elhai (unpublished)N2NH3O2

Слайд 34 Case of the Hidden Heterocyst
Strategy to find heterocyst

Case of the Hidden HeterocystStrategy to find heterocyst differentiation genes1. Use transposon mutagenesis

differentiation genes
1. Use transposon mutagenesis


Слайд 35
Case of the Hidden Heterocyst
Strategy to find heterocyst

Case of the Hidden HeterocystStrategy to find heterocyst differentiation genesNostoc genomeTransposon1.

differentiation genes


Nostoc genome
Transposon
1. Use transposon mutagenesis



to find a mutant

defective in heterocyst differentiation

Слайд 36
Case of the Hidden Heterocyst
Strategy to find heterocyst

Case of the Hidden HeterocystStrategy to find heterocyst differentiation genesNostoc genome2.

differentiation genes
Nostoc genome
2. Sequence out from transposon

AAGCTTGACCAAAAAGTTAAAACACTGACGGCAAATAATCAATGACTATCAGACAGAGAATCATCGTGCTGTCAGTAAAACCTCTGATTTCGATCTTTACCATAATTGTTATGTTGTAATGACTAACCAGACTATCTTTTACAGAGCTTCTGGTTAACACTTGTCTAATTAGACATTGATAATGTTTGTGGGGGTTGGTCATCAGGAATGGTAAATAGCAATTACCCTTCAGACTTTCCTATGAGACGCTCCGCCAACGAGCAGTGTCTCTTAAAGAACGTTATGAGCGCTCAGTTAACTTCAGAAATTCACGGCGGAAATCCATAGTTATTATTACTTATGACTAAAACAAAATTACTATGGCGGCTTGTTTAATATAGATTCTGTGTTCTGAGAAATGACTTTTAAAGTCCCACTAACTTTTTTCTCATCTATTGCTATATTTCGACTTTAAAACTTATAGTAGATGGCTTAATTCTCAAATAACAAACTCATTTTTAGTAGATATTTCATGCAAACTGAGGTTTTTAGTGATATTTTCCCCTTATTGAGTACAGCCACTCCACAAACCTTAGAATGGCTACTCAATATTGCAATTGATCATGAATATCCCACTGGTAGAGCAGTTTTAATGGAAGATGCCTGGGGTAATGCAGTTTATTTCGTTGTATCTGGATGGGTAAAAGTTCGGCGCACCTGTGGA
1. Use transposon

mutagenesis

to find a mutant defective in heterocyst differentiation


Слайд 37 Case of the Hidden Heterocyst
Strategy to find heterocyst

Case of the Hidden HeterocystStrategy to find heterocyst differentiation genesNostoc genome2.

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


Слайд 38 HetR
mutant - unable to make heterocysts

The spatially patterned

HetRmutant - unable to make heterocystsThe spatially patterned differentiation of heterocysts

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


Слайд 40
HetR
Genes needed for differentiation
Master regulator
Differentiation in cyanobacteria Integration of

HetRGenes needed for differentiationMaster regulatorDifferentiation in cyanobacteria Integration of signals through

signals through HetR
Position in filament
Position in cell cycle
Nitrogen deprivation

???? ??


Слайд 41 Promoter

Promoter

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


Слайд 42

NNNNNNNNNNNNNNNNNNATGNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNTACNNNNNNNNNNNNNNNN

hetR gene

5’-GTANNNTACNNNNNNNNNNTANNNTNNNNNNNNNN 3’-CATNNNATGNNNNNNNNNNATNNNANNNNNNNNNN





RNA Polymerase

Absence of fixed nitrogen

How might hetR be controlled?


Слайд 43

NNNNNNNNNNNNNNNNNNATGNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNTACNNNNNNNNNNNNNNNN

hetR gene

5’-GTANNNTACNNNNNNNNNNTANNNTNNNNNNNNNN 3’-CATNNNATGNNNNNNNNNNATNNNANNNNNNNNNN





RNA Polymerase

Absence of fixed nitrogen

How might hetR be controlled?

HetR protein

Transcription


Слайд 44 The key result of this experiment is that

The key result of this experiment is that all of the

all of the upstream controls of HetR expression can

be bypassed; expression of HetR alone suffices to turn on the differentiation pathway.

HetR overexpression


Слайд 46 PatS
overexpression of patS completely blocks heterocyst development
patS encode

PatSoverexpression of patS completely blocks heterocyst developmentpatS encode a 17- or

a 17- or 13-amino-acid peptide, is crucial for the

formation and maintenance of the normal heterocyst pattern

Слайд 47 Wild-type filaments (A) grown in BG-11 medium and

Wild-type filaments (A) grown in BG-11 medium and (B) after the

(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


Слайд 48 The exogenous addition of a pentapeptide corresponding to

The exogenous addition of a pentapeptide corresponding to the last five

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


Слайд 49 Přesně jako v modelu
z roku 1975

Přesně jako v modeluz roku 1975

The inhibition of neighboring cells by select differentiating cells (lateral inhibition) is an important mechanism of pattern formation in eukaryotic organisms.

Слайд 50
Because it takes ~20 hours for heterocysts to

Because it takes ~20 hours for heterocysts to mature and begin

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.

Слайд 51 + Nostoc punctiforme
Anthoceros punctatus.

+ Nostoc punctiformeAnthoceros punctatus.

Слайд 54 Další události nezbytné k aktivaci nif genů

Další události nezbytné k aktivaci nif genů

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