Summary of issues discussed during the second meeting of the “Sleeping
Beauty” project. (8-9/5/2006)
Present: P1 [Esther Lubzens, Ora Hadas, Nadav Denekamp (post-doc)]; P2 [Joan Cerdá,
Angèle Tingaud-Sequeua (post-doc)]; P3 [Stefan Hohmann, Avi Nahmani (PhD student),
Ivan Pirkov (PhD student), Cecilia Geijer (PhD student)]: P4 [Melody Clark, Jelena
Pilipović (PhD student)], P5 [Richard Reinhardt], P6 [Kristian Fog Nielsen].
Full and updated contact list is attached to this protocol
Notes were taken by Nadav Denekamp
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Agenda
Sleeping Beauty – Biennial Meeting, Novotel, Göteborg
May 7-9, 2006
Organized by Dr. Stefan Hohmann, Cecilia Geijer
Department of Cell and Molecular Biology/Microbiology
Göteborg University
Sweden
cecilia.geijer@gmm.gu.se
May 7: Meet on Sunday afternoon (15:00 Novotel Lobby) and go to Stefan's
place for a social event and informal discussions. Transfer will be arranged.
Return from Stefan’s place at 2000.
In case of direct transfer from airport:
Address: Norra häcksjöbäcksvägen 46, 44332 Lerum
Tel: +46 (0)302 17689, +46 (0)733547297
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May 8 – May 9 lunch time: presentations and discussions at a location to be
specified in Göteborg.
Presentations and Discussions
1. May 8: Presentations by partners and the co-ordinator. Partners will
present the progress, obstacles and plans for the following six months for
each part they participate in each one of the workpackages. Partner 6
(DTU) will present their institute and their role in the project.
2. May 9: Each leader of a work package will summarise the progress in
their workpackage and highlight obstacles and plans for the forthcoming 6
months. Any deviation in the workplan or the timetable will be discussed.
Discussions will be held on the flow of samples and their analyses by
Partner 5 (MPI-MG) and Partner 6 (DTU). Discussions will be held on
topics raised by partners. Discussions will be initiated on the Dormancy
workshop in 2008.
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1.1 Agenda for Monday May 8, 2006
0900 - 0915
Welcome
Stefan
Hohmann
(P3)
0915 – 0945
Cynanobacteria akinetes
Ora Hadas, Assaf
Sukenik (P1-KLL)
0945 - 1015
Yeast spores
Stefan
Hohmann
(P3)
1015 - 1045
Coffee Break
1045 - 1115
Rotifer resting eggs
Esther Lubzens (P1NIO)
1115 - 1145
Arctic Springtails
Roger
Worland,
Melody Clark (P4)
1145 - 1215
Killifish eggs
Joan Cerdá (P2)
1215 -1245
Discussion
Esther Lubzens
1245 - 1400
Lunch
1400 - 1530
Management and coordination of project
Stefan
data – Workpackage 5
and Melody Clark
DTU and metabolomics
Kristian Fog Nielsen
1530 - 1600
Hohmann
(P6)
1600 - 1630
Molecular and Proteomic tools
Richard
Reinhardt
(P5)
1630- 1700
Coffee break
1700 – 1800
Discussion on tools and General Discussion
Richard
Reinhardt,
Kristian Fog Nielsen
and Esther Lubzens
1900
Dinner
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1.2 Agenda for Tuesday, May 9, 2006
0830 -0845
Inroduction into discussion on SB Esther Lubzens
workplan and Workpackage 0
0845 - 0915
Workpackage 1 – Coordinators: Ora Hadas, Assaf Sukenik (P1Esther Lubzens & Ora Hadas
KLL), Esther Lubzens (P1-NIO),
Stefan Hohmann (P3), Richard
Reinhardt (P5)
0915 - 0945
Workpackage 2 –
Coordinator: Stefan Hohmann
Ora Hadas, Assaf Sukenik (P1KLL), Esther Lubzens (P1-NIO),
Stefan Hohmann (P3), Richard
Reinhardt (P5)
0945 - 1015
Coffee Break
1015 – 1045
Workpackage 3 –
Coordinator: Joan Cerdá
Joan Cerdá (P2) Roger Worland
(P4),
Melody
Clark
(P4),
Richard Reinhardt (P5)
1045 - 1115
Workpackage 4 –
Coordinator: Joan Cerdá
Esther Lubzens (P1-NIO), Joan
Cerdá (P2), Stefan Hohmann
(P3), Richard Reinhardt (P5)
1115 - 1145
Workpackage 5 –
Conclusions from previous day
Coordinators: Stefan Hohmann & All Partners
Melody Clark
1145 -1215
Workpackage 6 –
All partners
Coordinator: Esther Lubzens
1215 -1230
Summing up
1230
Lunch
Esther Lubzens
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Presentations made by partners
The presentations will be available in PDF format in the project web site.
(http://www.gmm.gu.se/sleeping). The presentations included reports on achievements,
deliverables and plans until M12 and for achieving milestones.
1. Cyanobacteria Dormancy Forms in an Aquatic environment [Ora Hadas]
2. Yeast spores
2.1. Sporulation in buddingyeast S. cerevisiae and the story of yeast aquaporin Aqy1
[Cecilia Geijer]
2.2. Mechanisms of Dormancy and Germination of The Baker’s Yeast S. cerevisiae
Spore [Ivan Perkov]
3. Rotifer resting eggs [Nadav Denekamp and Esther Lubzens]
4. Arctic Sprintails [Melody Clark]
5. Killifish eggs [Angèle Tingaud-Sequeua]
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Management and coordination of project data - WP5 [Stefan hohmann and
Melody Clark]
3.1
Microarray experiments design and data analysis.
Support for experiments design for those who do not have experience with microarray
experiments (P1 and P2) will be given by P3 (i.e. Avi). Analysis of the microarray data
will be done by each partner. A workshop on microarray analysis will be given by P3.
3.2
Analysis of the results
Further analysis of the results (after data analysis) - discovery of common and divergent
pathways for dormant stages, dormancy and desiccation resistance will be co-ordinated
by P3 and P4 (Melody).
It was suggested to build a platform to examine the effect of different conditions and/or
stages within the life cycle on the expression of genes, however such platform maybe too
complicated.
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3.3
Common gene bank
It was agreed that when the EST data (sequences and annotations) are available they will
be linked to the project web site (privet area). The web site will contain a search and
blast utilities within the ESTs of all partners.
The issue of keeping the data confident was brought up. It was agreed that if one wishes
to share data from the project with a collaborator he may share his/her own data but with
permission of all partners. The main idea is to give the partners the privilege to use first
knowledge that was generated from the project.
When there are results of all the microarray experiments, there will be another discussion
on the combination of the results. The discussion will take place in the end of the second
year during the annual meeting.
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DTU and metabolomics [Kristian F. Nielsen]
The platforms offered by DTU are described at Kisrstian’s presentation, which will be
available in the project website (PDF format).
Partners should supply Kristian the following information: sample matrix (needed for
sample preparation), amount available (mg, gram…), special metabolites and the number
of samples.
Partners will send samples to Kristian during the following months so that he will be able
to develop methods for analysis. Please send an email to Kristian before sending samples.
Kristian’s contact information is listed in the updated contact list attached to the protocol.
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5.1
Molecular and proteomic tools [Richard]
cDNA libraries and ESTs
Until now P2(Joan) and P4(Melody) sent RNA samples to P5. P1-KLL (Ora) sent
samples for genome sequencing but they need to be sent again. Samples fromP1-NIO
(Esther) will be sent during the next 3 months.
cDNA libraries will be constructed for different condition and/or stages. cDNA from all
the libraries will be pooled into one normalized library.
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Until the next meeting each partner should tell Richard how many additional ESTs
(beyond the 15k covered by the project) are requested.
The original list for Richard:
 56K ESTs
 ~14 libraries
 Microarrays: 3,000 genes/array and 40 experimetns
The current list:
 P1(Ora): Whole genome sequencing in exchange of 3 libraries and 14,000 ESTs.
 P1(Esther): About 6 libraries to be normalized into 1 and 16,000 ESTs
 P2(Joan): About 6 libraries to be normalized into 1 and 15,000 ESTs
 P4(Melody): 3(?) normalized libraries, no microarrays.
The ESTs should be prepared until the end of 2006.
5.2
Microarrays
Microarrays services of P5 are needed for P1 and P2. P4 (Melody) will make their own
slides.
Printing, hybridization and scanning of the first ten slides are covered within the project
budget. Richard will supply the costs for additional hybridization experiments until the
next meeting. The slides will be printed only once, thus, requests for additional slides
should be made in advance. The available amount of cDNA may limit the number of
slides. Every slide will contain 3,000 genes (including replicate spots). The slides may
contain genes from one treatment or genes from two treatments (1,500 genes per
treatment). This issue should be discussed in the next meeting.
Hybridization and scanning of additional slides may be done in Berlin or by each partner.
It was suggested to organize a workshop on hybridization experiments for those who
whish to perform the experiments by themselves.
5.3
Proteome analysis
Proteome analysis services of P5 are needed for P1, P2, P4. P3 (Stefan) will perform the
proteome analysis for yeasts.
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Proteome profiling: 2D gels and analysis of ~400 spots.
2D gels analysis needs optimization therefore partners are requested to send a picture of a
2D gel of their samples to Richard or to send samples just for optimization. The amount
of protein needed for 2D gel is 50-100 g per lane.
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6.1
Discussions on the workpackages.
WP0 – management
6.1.1 Next meetings
M12: meeting in Cambridge 16-18/10/2006.
M18: Copenhagen DTU (May 2007).
M24: Israel NIO+KLL. The place of the meeting will be decided according to a
questionnaire that will be sent by email (October 2007).
M30: The workshop on dormancy and the biennial meeting in Berlin (May 2008).
M36: Barcelona (October, 2008).
6.1.2 Contract between the partners
Esther will send a copy of a proposed contract between the partners.
6.1.3 Project website
The website will be available soon. It was agreed not to put manuscript on the website
but just the links to the publisher site.
6.2
WP1: Studies on mechanisms of establishments of dormant stages
D4: Optimal conditions for the induction of akinetes in cyanobacteria, asexualy and
sexually reproducing rotifers and their resting eggs (M9) – accomplished.
D5: cDNA libraries , EST sequencing and database construction – in progress (M18).
RNA samples from P1.NIO will be sent to P5 during the next 3 months.
D6: Pattern of global gene expression during formation of akinetes, rotifer resting eggs
by micorarray anaylysis (M27) – will start after D4 is accomplished.
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D7: Quantitative analysis of expression of specific genes by QPCR (M35) – will start
after D6 is accomplished.
D8: Proteins and metabolites identification to be associated with formation of dormant
stages within dormant forms (M30) – samples for metabolomics and proteomics
calibration will be sent during the next months.
D9: Genes, proteins and metabolites associated with the dormant stages of yeast spores
(M36) – on work.
6.3
WP2: Studies on the mechanisms of arousal from dormant stage
D10: Metabolic pathways associated with germination in akinetes and of vegetative
reproducing cyanobacteria (M12) – in progress.
D11: Pattern of global gene expression during exit from dormancy of akinetes , resting
eggs and spores by microarray analysis (M35) – will start when D5 is finished.
D12: Quantitative expriession levels of specific genes by QPCR (M35) – will start after
D11 is finished.
D13: Protein and metabolites identified to be associated with resume metabolic activities
in akinetes and during embryonic development in resting eggs and asexual rotifer eggs
(M30) - samples for metabolomics and proteomics calibration will be sent during the next
months.
D14: Genes, proteins and metabolites associated with yeast spores germination (M36) –
on work.
6.4
WP3: Desiccation and revival
D15: Identification of cooling rate that elicits trehalose synthesis in Arctic srpringtails
(M12) – almost finished
D16: Identification and characterization of killifish embryo stages resistant to air
exposure (M12) – almost finished.
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D17: Microarray analysis of global genes expression during desiccation resistance in
sprintails and air exposure in killifish embryos – in progress, RNA samples were sent to
P5. Results of ESTs of the killifish embryos should be available until October 2006.
D18: ISH and quantitave mRNA expression levels of specific genes by Real-time PCR
(M35) - QPCR for the genes HSP70, HIF and HIF2are in progress.
D19: Protein and metabolic profiles associated with desiccated springtails and airexposed killifish embryos (M30) – samples for metabolomics and proteomics calibration
will be sent during the next months.
6.5
WP4 - Engineering/genetic testing strategies
Not started yet
6.6
WP5- Discovery of common divergent pathways for dormant stages,
dormancy and desiccation resistance.
Not started yet.
6.7
WP6 – Dissemination of results.
6.7.1 A workshop on dormancy.
Place and time
Berlin (MPI), May 2008
Suggested titles
 Sleeping beauties: Dormancy in organism
 Molecular, proteomic and metabolomic aspects of dormancy
 Dormancy and desiccation: molecular, proteomic and metabolomic aspects
 Genes, proteins and compounds associated with dormancy
 Dormancy: genes proteins and compound
Suggested sessions
 Dormancy - mechanisms associated with onset of dormancy
 Mechanisms associated with “revival” (germination; hatching; renewal of metabolism)
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 Hibernation
 Signal transduction
 Compound in dormant forms
Duration and agenda
Duration of the workshop should be 2-3 days. The proposed agenda for each day is:
Day1
Day 2
Day 3
Session 1:
09:00-10:30
Session 2:
11:00-12:30
Session 3:
14:00-15:30
Session 4:
16:00-18:00 (including round table discussion)
Session 5:
09:00-10:30
Session 6:
11:00-12:30
Session 7:
Posters?
Session 8:
16:00-18:00 (including round table discussion)
Session 9:
09:00-10:30
Session 10:
11:00-12:30
Session 11:
14:00-15:30
Session 12:
16:00-18:00
Festive Dinner Day 3
Total: 18 hours (36-45 oral presentations)
Invited speakers:~12 (~10,000 Euro) or 24 (~20,000 Euro)
Students :6,000 Euro
Addition expenses: ~5,000 Euro
Total: ~23,000 or 35,000 Euro
Scientific organizing committee
Chairpersons are to be decided. Proposed persons for the organizing:
 Microorganisms: Stefan? ,W. Hess?
 Plants: Derek Bewley, Henk Hillhorst
 Algae and macroalgae : Catherine Boyen?
 Invertebrates: Clegg? Tunnacliffe? Esther?
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 Insects: Roger?
 Vertebrates: Ken Storey? Joan?
 Compounds?
Each session will be organized by 1 person from SB with a leading scientist in the field.
Funding
The workshop is partially budgeted by the project. It is suggested to apply to EMBO for
additional funding. The deadline for submitting the proposal is February, 2007. The
proposal should be submitted by one of the European partners and not by Esther because
Israel is not a member of EMBO.
6.7.2 Publication of a book on dormancy
Stefan will take care for publishing the book in a series that he is editing. The process
will take about 2 years. It is suggested that people who will participate in the workshop
will be invited to write reviews in the book.
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Issues to be discussed in the next meeting
Partners are requested to send the abstract of their report to Esther a month before
the meeting.
7.1
ESTs and micorarrays
 Partners requests for additional ESTs and microarray slided.
 The cost of additional ESTs and microarray experiments will be supplied by Richard.
 Workshop on microarray experiments.
 Workshop on microarray analysis.
7.2
Workshop on dormancy
 Title
 Sessions
 Participants
 Organizing committee
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