EPIGENETIKA MB150P85

EPIGENETIKA MB150P85

Přírodovědecká fakulta UK

Petr Svoboda

mail: [email protected]: 241063147

mailto:[email protected]

DNA METHYLATION I(DNA METHYLATION AND ITS DETECTION)

EPIGENETIKA

Epigenetika se zabývá přenosem vlastností (informací), které nejsou uložené v sekvenci DNA.

Tyto informace jsou přenášené především v:

- struktuře a modifikacích chromatinu

- chemických modifikacích DNA

- RNA molekulách

Bacteria - protection against RE

Eukaryota - numerous effects

Bacteria - protection against RE

Bacteria - wide range of functions

Protista, Plantae, (Mammalia?)

See Ratel 2006

-CcGG- -GGcC-

all sites * Hemophilus parainfluenzae

-cCGG- -GGCc-

all sites *Moricardia sp.

-GaTC- -CTaG-

all sites (dam)Escherichia coli

-Cc(A/T)GG- -GG(T/A)cC-

all sites (dcm)Escherichia coli

-cG- -Gc-

some sites **mammals, some fungi (Neurospora) and plants

-cNG- -GNc-

some sites **plants

An example of a modified restriction enzyme recognition site. These sites are usually modified in organisms with the corresponding restriction activity.

** Fraction of CG dinucleotides or CNG trinucleotides varies with species and, to a lesser extent, with tissue.

Specific nucleotides are modified

http://opbs.okstate.edu/~melcher/mg/MGW3/MG3134.html

DNA methylation

http://www.med.ufl.edu/biochem/keithr/research.html

5-methyl cytosine methylation

Mammalian DNA methyltransferases

Maintenance DNA methylation

De novo DNA methylation

Ignore

HW: Why?

Robertson 2002

Methods to study DNA methylation

Global methylation analysis

HPLC (complete hydrolysis, AP)

TLC (complete hydrolysis, AP, 32P labeled)

Sequence-specific methylation analysis

Methylation sensitive restriction enzymes - a number of methods

Bisulfite sequencing

MeDIP

For more details, see Oakeley, 1999

Bisulfite sequencing

From Oakeley, 1999

http://www.methods.info/Methods/DNA_methylation/Bisulphite_sequencing.html

dsDNA is resistant to conversion!

HW:Why isn’t 5mC

converted?

Classical protocol (bisulfite protocol.doc)

- starting material: cells or DNA up to 200 ng- extremely sensitive (100 DNA molecules)- based on agarose embedding

Drawbacks:- time consuming (approx. 10-11 hours)- low throughput (typically up to 8 samples/run)- low yield (200 ng max/reaction)

Epitect Bisulfite Sequencing Kit(Qiagen)

http://www1.qiagen.com/Products/Epigenetics/Epitect/EpitectBisulfiteKit.aspx?ShowInfo=1

Epitect

- starting material: DNA 1 ng - 2 g- sensititvity OK for most applications- faster (cca 6 hours), throughput OK

Whatever …

The critical component are primers!!

AAAn

ORF1 ORF25’ UTREN RT

~6 kb

- active, autonomous, non-LTR class

- retrotransposition in cis

- the most abundant retrotransposon in mammalian genomes

- 4-500 000 insertion in the human genome

- ~100 full length intact elements

- typically silenced in somatic cells (hypermethylation)

Model case:L1 promoter methylation analysis

>L1 5’ UTRGGGGGGAGGAGCCAAGATGGCCGAATAGGAACAGCTCCGGTCTACAGCTCCCAGCGTGAGCGACGCAGAAGACGGTGATTTCTGCATTTCCATCTGAGGTACCGGGTTCATCTCACTAGGGAGTGCCAGACAGTGGGCGCAGGCCAGTGTGTGTGCGCACCGTGCGCGAGCCGAAGCAGGGCGAGGCATTGCCTCACCTGGGAAGCGCAAGGGGTCAGGGAGTTCCCTTTCCGAGTCAAAGAAAGGGGTGACGGACGCACCTGGAAAATCGGGTCACTCCCACCCGAATATTGCGCTTTTCAGACCGGCTTAAGAAACGGCGCACCACGAGACTATATCCCACACCTGGCTCGGAGGGTCCTACGCCCACGGAATCTCGCTGATTGCTAGCACAGCAGTCTGAGATCAAACTGCAAGGCGGCAACGAGGCTGGGGGAGGGGCGCCCGCCATTGCCCAGGCTTGCTTAGGTAAACAAAGCAGCAGGGAAGCTCGAACTGGGTGGAGCCCACCACAGCTCAAGGAGGCCTGCCTGCCTCTGTAGGCTCCACCTCTGGGGGCAGGGCACAGACAAACAAAAAGACAGCAGTAACCTCTGCAGACTTAAGTGTCCCTGTCTGACAGCTTTGAAGAGAGCAGTGGTTCTCCCAGCACGCAGCTGGAGATCTGAGAACGGGCAGACTGCCTCCTCAAGTGGGTCCCTGACCCCTGACCCCCGAGCAGCCTAACTGGGAGGCACCCCCCAGCAGGGGCACACTGACACCTCACACGGCAGGGTATTCCAACAGACCTGCAGCTGAGGGTCCTGTCTGTTAGAAGGAAAACTAACAACCAGAAAGGACATCTACACCGAAAACCCATCTGTACATCACCATCATCAAAGACCAAAAGTAGATAAAACCACAAAG

1) Find your sequence – NCBI Genbank and Pubmed

BISULFITE SEQUENCING STEP BY STEP


>L1 5’ UTRGGGGGGAGGAGCCAAGATGGCCGAATAGGAACAGCTCCGGTCTACAGCTCCCAGCGTGAGCGACGCAGAAGACGGTGATTTCTGCATTTCCATCTGAGGTACCGGGTTCATCTCACTAGGGAGTGCCAGACAGTGGGCGCAGGCCAGTGTGTGTGCGCACCGTGCGCGAGCCGAAGCAGGGCGAGGCATTGCCTCACCTGGGAAGCGCAAGGGGTCAGGGAGTTCCCTTTCCGAGTCAAAGAAAGGGGTGACGGACGCACCTGGAAAATCGGGTCACTCCCACCCGAATATTGCGCTTTTCAGACCGGCTTAAGAAACGGCGCACCACGAGACTATATCCCACACCTGGCTCGGAGGGTCCTACGCCCACGGAATCTCGCTGATTGCTAGCACAGCAGTCTGAGATCAAACTGCAAGGCGGCAACGAGGCTGGGGGAGGGGCGCCCGCCATTGCCCAGGCTTGCTTAGGTAAACAAAGCAGCAGGGAAGCTCGAACTGGGTGGAGCCCACCACAGCTCAAGGAGGCCTGCCTGCCTCTGTAGGCTCCACCTCTGGGGGCAGGGCACAGACAAACAAAAAGACAGCAGTAACCTCTGCAGACTTAAGTGTCCCTGTCTGACAGCTTTGAAGAGAGCAGTGGTTCTCCCAGCACGCAGCTGGAGATCTGAGAACGGGCAGACTGCCTCCTCAAGTGGGTCCCTGACCCCTGACCCCCGAGCAGCCTAACTGGGAGGCACCCCCCAGCAGGGGCACACTGACACCTCACACGGCAGGGTATTCCAACAGACCTGCAGCTGAGGGTCCTGTCTGTTAGAAGGAAAACTAACAACCAGAAAGGACATCTACACCGAAAACCCATCTGTACATCACCATCATCAAAGACCAAAAGTAGATAAAACCACAAAG

1) Find your sequence

HW:look at CpG density of: EGFP, Bluescript, Actin promoter and transcribed region, IAP LTR, L1 ORF2


>L1 5’ UTRGGGGGGAGGAGCCAAGATGGCXYAATAGGAACAGCTCXYGTCTACAGCTCCCAGXYTGAGXYAXYCAGAAGAXYGTGATTTCTGCATTTCCATCTGAGGTACXYGGTTCATCTCACTAGGGAGTGCCAGACAGTGGGXYCAGGCCAGTGTGTGTGXYCACXYTGXYXYAGCXYAAGCAGGGXYAGGCATTGCCTCACCTGGGAAGXYCAAGGGGTCAGGGAGTTCCCTTTCXYAGTCAAAGAAAGGGGTGAXYGAXYCACCTGGAAAATXYGGTCACTCCCACCXYAATATTGXYCTTTTCAGACXYGCTTAAGAAAXYGXYCACCAXYAGACTATATCCCACACCTGGCTXYGAGGGTCCTAXYCCCAXYGAATCTXYCTGATTGCTAGCACAGCAGTCTGAGATCAAACTGCAAGGXYGCAAXYAGGCTGGGGGAGGGGXYCCXYCCATTGCCCAGGCTTGCTTAGGTAAACAAAGCAGCAGGGAAGCTXYAACTGGGTGGAGCCCACCACAGCTCAAGGAGGCCTGCCTGCCTCTGTAGGCTCCACCTCTGGGGGCAGGGCACAGACAAACAAAAAGACAGCAGTAACCTCTGCAGACTTAAGTGTCCCTGTCTGACAGCTTTGAAGAGAGCAGTGGTTCTCCCAGCAXYCAGCTGGAGATCTGAGAAXYGGCAGACTGCCTCCTCAAGTGGGTCCCTGACCCCTGACCCCXYAGCAGCCTAACTGGGAGGCACCCCCCAGCAGGGGCACACTGACACCTCACAXYGCAGGGTATTCCAACAGACCTGCAGCTGAGGGTCCTGTCTGTTAGAAGGAAAACTAACAACCAGAAAGGACATCTACACXYAAAACCCATCTGTACATCACCATCATCAAAGACCAAAAGTAGATAAAACCACAAAG

2) Word -> REPLACE CG WITH XY


>L1 5’ UTRGGGGGGAGGAGTTAAGATGGTXYAATAGGAATAGTTTXYGTTTATAGTTTTTAGXYTGAGXYAXYTAGAAGAXYGTGATTTTTGTATTTTTATTTGAGGTATXYGGTTTATTTTATTAGGGAGTGTTAGATAGTGGGXYTAGGTTAGTGTGTGTGXYTATXYTGXYXYAGTXYAAGTAGGGXYAGGTATTGTTTTATTTGGGAAGXYTAAGGGGTTAGGGAGTTTTTTTTTXYAGTTAAAGAAAGGGGTGAXYGAXYTATTTGGAAAATXYGGTTATTTTTATTXYAATATTGXYTTTTTTAGATXYGTTTAAGAAAXYGXYTATTAXYAGATTATATTTTATATTTGGTTXYGAGGGTTTTAXYTTTAXYGAATTTXYTTGATTGTTAGTATAGTAGTTTGAGATTAAATTGTAAGGXYGTAAXYAGGTTGGGGGAGGGGXYTTXYTTATTGTTTAGGTTTGTTTAGGTAAATAAAGTAGTAGGGAAGTTXYAATTGGGTGGAGTTTATTATAGTTTAAGGAGGTTTGTTTGTTTTTGTAGGTTTTATTTTTGGGGGTAGGGTATAGATAAATAAAAAGATAGTAGTAATTTTTGTAGATTTAAGTGTTTTTGTTTGATAGTTTTGAAGAGAGTAGTGGTTTTTTTAGTAXYTAGTTGGAGATTTGAGAAXYGGTAGATTGTTTTTTTAAGTGGGTTTTTGATTTTTGATTTTXYAGTAGTTTAATTGGGAGGTATTTTTTAGTAGGGGTATATTGATATTTTATAXYGTAGGGTATTTTAATAGATTTGTAGTTGAGGGTTTTGTTTGTTAGAAGGAAAATTAATAATTAGAAAGGATATTTATATXYAAAATTTATTTGTATATTATTATTATTAAAGATTAAAAGTAGATAAAATTATAAAG

3) Word -> REPLACE C WITH T


>L1 5’ UTRGGGGGGAGGAGTTAAGATGGTCGAATAGGAATAGTTTCGGTTTATAGTTTTTAGCGTGAGCGACGTAGAAGACGGTGATTTTTGTATTTTTATTTGAGGTATCGGGTTTATTTTATTAGGGAGTGTTAGATAGTGGGCGTAGGTTAGTGTGTGTGCGTATCGTGCGCGAGTCGAAGTAGGGCGAGGTATTGTTTTATTTGGGAAGCGTAAGGGGTTAGGGAGTTTTTTTTTCGAGTTAAAGAAAGGGGTGACGGACGTATTTGGAAAATCGGGTTATTTTTATTCGAATATTGCGTTTTTTAGATCGGTTTAAGAAACGGCGTATTACGAGATTATATTTTATATTTGGTTCGGAGGGTTTTACGTTTACGGAATTTCGTTGATTGTTAGTATAGTAGTTTGAGATTAAATTGTAAGGCGGTAACGAGGTTGGGGGAGGGGCGTTCGTTATTGTTTAGGTTTGTTTAGGTAAATAAAGTAGTAGGGAAGTTCGAATTGGGTGGAGTTTATTATAGTTTAAGGAGGTTTGTTTGTTTTTGTAGGTTTTATTTTTGGGGGTAGGGTATAGATAAATAAAAAGATAGTAGTAATTTTTGTAGATTTAAGTGTTTTTGTTTGATAGTTTTGAAGAGAGTAGTGGTTTTTTTAGTACGTAGTTGGAGATTTGAGAACGGGTAGATTGTTTTTTTAAGTGGGTTTTTGATTTTTGATTTTCGAGTAGTTTAATTGGGAGGTATTTTTTAGTAGGGGTATATTGATATTTTATACGGTAGGGTATTTTAATAGATTTGTAGTTGAGGGTTTTGTTTGTTAGAAGGAAAATTAATAATTAGAAAGGATATTTATATCGAAAATTTATTTGTATATTATTATTATTAAAGATTAAAAGTAGATAAAATTATAAAG

4) Word -> REPLACE XY WITH CG


>L1 5’ UTRGGGGGGAGGAGTTAAGATGGTCGAATAGGAATAGTTTCGGTTTATAGTTTTTAGCGTGAGCGACGTAGAAGACGGTGATTTTTGTATTTTTATTTGAGGTATCGGGTTTATTTTATTAGGGAGTGTTAGATAGTGGGCGTAGGTTAGTGTGTGTGCGTATCGTGCGCGAGTCGAAGTAGGGCGAGGTATTGTTTTATTTGGGAAGCGTAAGGGGTTAGGGAGTTTTTTTTTCGAGTTAAAGAAAGGGGTGACGGACGTATTTGGAAAATCGGGTTATTTTTATTCGAATATTGCGTTTTTTAGATCGGTTTAAGAAACGGCGTATTACGAGATTATATTTTATATTTGGTTCGGAGGGTTTTACGTTTACGGAATTTCGTTGATTGTTAGTATAGTAGTTTGAGATTAAATTGTAAGGCGGTAACGAGGTTGGGGGAGGGGCGTTCGTTATTGTTTAGGTTTGTTTAGGTAAATAAAGTAGTAGGGAAGTTCGAATTGGGTGGAGTTTATTATAGTTTAAGGAGGTTTGTTTGTTTTTGTAGGTTTTATTTTTGGGGGTAGGGTATAGATAAATAAAAAGATAGTAGTAATTTTTGTAGATTTAAGTGTTTTTGTTTGATAGTTTTGAAGAGAGTAGTGGTTTTTTTAGTACGTAGTTGGAGATTTGAGAACGGGTAGATTGTTTTTTTAAGTGGGTTTTTGATTTTTGATTTTCGAGTAGTTTAATTGGGAGGTATTTTTTAGTAGGGGTATATTGATATTTTATACGGTAGGGTATTTTAATAGATTTGTAGTTGAGGGTTTTGTTTGTTAGAAGGAAAATTAATAATTAGAAAGGATATTTATATCGAAAATTTATTTGTATATTATTATTATTAAAGATTAAAAGTAGATAAAATTATAAAG

5) Design primers- size typically up to 500 bp, ideally around 300 bp

- avoid low complexity sequences- Tm around 55oC - select the right region!

TTTT doesn’t matter

3’ End dG should be above -13.0(calculated from the last 7 nucleotides)

- ignore all dimers with positive dG- only primers with negative dG <-1.5 should concern you- combination of low negative dG and perfect basepairing at the 3’ end is the worst combination

Duplex analysis

Tips how to improve amplification

HOT START PCR

-add Taq pol only after denaturation step- use hot start Taq pol, e.g. Amplitaq GOLD from Perkin Elmer/ABI

- Amplitaq GOLD requires the intital denaturation step for 10-15 min at 95 oC

TOUCHDOWN PCR

94 oC for 15 min

94 oC for 30 sec62->55 oC for 30 sec72 oC for 1 min

94 oC for 30 sec55 oC for 30 sec72 oC for 1 min

72 oC for 15-20 min

14 cycles0.5 oC down/cycle

36 cycles

EXTREMELY SENSITIVE TO

CONTAMINATIONS !!!


5) Run PCR

6) cut 7) Gel Extraction(Qiagen)

8) TOPO TA II cloning(Invitrogen)

9) Miniprep(Qiagen)

10) Sequencing with SP6 primer

Restriction digest- cheap and fast- less information (up to a few CpGs)- not good for polymorphic sequences

Pyrosequencing http://www.pyrosequencing.com/

- short read (30nt)- quantitative ratio of polymorphic nucleotides- good for one sequence analyzed from many samples

Direction of sequencing is important!

-GT-rich regions are more difficult to sequence- cause problems especially with products >300 bp

400

700

The same PCR product (amount, purity …) but different strands sequenced

BioEdit –it’s good and it’s free!http://www.mbio.ncsu.edu/BioEdit/bioedit.html

Vector NTI is good but f*cking expensive

52 58 61 70

analysis of individual elements

single-locus analysis

AAAn


L1 p

ool

L1 6

p21

L1 6

p22

L1 6

q16

L1 8

q24

L1 X

p22

L1 X

q22

? ? ?

femaleundifferentiated

hES sample

How would you explain it?How would you test your explanation?

AAAn


8q24

Xp22

HOMEWORK

Oct-4 promoter analysis-PCR cloned in pCR II-sequenced with SP6

DNA METHYLATION II(EFFECTS OF DNA METHYLATION)

EUMETAZOA

PSEUDOCOELOMATES

COELOMATESPROTOSTOMES

DEUTEROSTOMESECHINODERMATA

CHORDATA

AMPHIBIA

NEMATODA

PISCES

MAMMALIA

ARTHROPODA

>350 MYA

>400 MYA

>600 MYA

Mus

Xenopus

Danio

Strongylocentrotus

Drosophila

Caenorhabditis

- a number of conserved genes - common basic cell types - highly variable DNA methylation - different development - conserved histone modifications- different sex determination- different epigenetic mechanisms

DNA METHYLATION ACROSS PHYLA

Jabbari 2004

-genomes carrying CpG methylation show lower frequencies of CpG

EXTENSIVE DNA METHYLATION LEAVES TRACES ….

http://www.chemsoc.org/chembytes/ezine/2001/pufulete_mar01.htm

C-T CONVERSION

0.1-0.2% of the cytosines methylated

Drosophila

DNA methylation in the zebrafish

DNA METHYLATION DISTRIBUTION- trends and exceptions

repetitive sequences

- noncoding tandem repeats (satellites) hypermethylated- coding tandem repeats - rDNA variable- interspersed elements - L1, IAP, Alu hypermethylated- telomeric repeats hypermethylated

unique sequences

- promoter - active genes +/- unmethylated- inactive genes variable

- “gene body” methylated

upstream of the UCE UCE core p.

dim-H3K9(inactive)

H4Ac(active)

HEK-293total DNA

rDNA

- pol I transcription- tandemly arrayed on five pairs of human acrocentric chromosomes- ~400 copies per haploid genome- typically half active, half inactive (all active in the oocyte)

Methylationhypo hyper

transformed cell lines 50% 50%primary human cells 100% 0%primary mouse cells 100% 0%undiff. ES cells 100% 0%diff. ES cells 100% 0%

130 bp repeats UCE Core

Annotation of UCE and CORE sequences based on Heix and Grummt , 1995

400 bp

human

mouse

- different CpG density between closely related species- gene activity does not correlate with methylation in both species- only inactive genes in transformed lines acquire methylation- sometimes, methylation is not very informative

Kim

et

al.

BM

C C

an

cer

20

06

6:1

80

CpG islands

CpG island is a region least 200 bp long and with a GC

percentage that is greater than 50% and with CpG frequency

that is greater than 6% (genome average is 1%).

Found in and near approximately 40% of

promoters of mammalian genes (about 70% in human promoters). A “typical” CpG island is 300-3000 bp long. The CpG sites in the CpG islands of promoters are typically unmethylated if

genes are expressed. This observation led to the

speculation that methylation of CpG sites in the promoter of a

gene may inhibit the expression of a gene.

http://www.biomedcentral.com/1471-2407/6/180/figure/F2

PROMOTERS OF INACTIVE GENES

- hundreds of papers with contradictory data- methylation correlates with inactivity … but that’s it ...

CpG poor promoters - hypermethylated regardless of activitystrong CpG island promoters - hypomethylated regardless of activityweak CpG island promoters - distinct …testis-specific promoters - methylated in somatic cells


Maintenance methylation - DNMT1

substrate: hemimethylated DNAfunction: restoration of DNA methylation after replication

De novo methylation - DNMT3a, 3b (3l)

substrate: unmethylated DNAfunction: establishemnt of new DNA methylation patterns

Li 2002


Maintenance DNA methylation

De novo DNA methylation

Ignore

HW: Why?

Robertson 2002

Setting up and interpreting the mark …

Klose 2006

DNA Methyl Binding Proteins MBDs

http://www.wcb.ed.ac.uk/bird.htm

http://homepages.ed.ac.uk/dmac/Bird_Lab/birdlab.html

MBDs

Klose 2006

Hendrich 2003

MBD1

MBD2

MBD3

MBD4

MECP2

KO MUTATIONSCHARACTERIZATION

-transcriptional repressor- X-linked-able to bind a single methyl-CpG-binds tightly to chromosomes, pericentromeric heterochromatin- associates with Sin3A HDAC complex and BRM complex- expression correlates with maturation of the neural system

-mutant ES cells grow and can differentiate (Tate et al., 1996)-essential for embryonic development (Tate et al., 1996)-not essential for embryonic development (Chen et al. 2001)

Rett syndrome- various mutations- only in females (lethal in m.)

Angelmann syndrome?Autism?

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM

-transcriptional repressor from the MeCP1 complex- also described as demethylase - but nobody reproduced that!-MBD2/ NuRD complex, HDACs-MBD2 recruiting the NURD complex to methylated DNA?

-glycosylase domain, DNA repair- interacts with mismatch repair protein MLH

primary microsatellite-instability (MSI) tumors

-Mbd4 -/- mice have a 3-fold increase in the frequency of C-to-T transitions at CpG sites (Millar et al. 2002)

-transcriptional repressor from the MeCP1 complex-requires more than a single methyl-CpG-in a complex with HDAC3-interacts with the Suv39h1-HP1 heterochromatic complex

-does not bind methylated DNA-MBD3/ NuRD complex (distinct from MBD2 NURD)-MBD3 an integral part of the NURD complex?

-Mbd3 -/- ES cells are viable but fail to form a stable NuRD complex, differentiation defect, LIF-independent self-renewal. - Mbd3-/- mice die during early embryogenesis

Kaiso -lacks MBD domain-recognizes 5mC in CGCG through zinc-finger domains- transcriptional repressor

Mbd-/- mice have deficits in adult neurogenesis and hippocampal function (Zhao et al., 2003)

Autism?

Mbd2-/- mice are viable and fertile! Maternal behavior of Mbd2-/- mice is defective (Hendrich et al. 2001)

Kaiso -/-t mice show resistance to intestinal cancer

Klose 2006

- when it is associated with transcriptional repression, there are usually also chromatin modifications consistent with the pattern

- information about DNA sequence methylation without information about location (promoter, intergenic, intronic, exonic …) and expression has low information content

- methylated promoter does not automatically belong to an inactive gene while unmethylated promoter does not automatically belong to an active gene

- it’s OK to say that DNA methylation is typically associated with transcriptional repression

- be open minded and expect the unexpected ...

A few final notes regarding DNA methylation

- inhibition of DNMT1, - - replication dependent

DNA DEMETHYLATION

PASSIVE

- replication independent- clearly demonstrated in some cases- unknown mechanism, unknown demethylase. DNA repair/glycosylase?

ACTIVE

- DNA methylation is stable but reversible

CpG methylation during development

Adapted from Mayer et al. (2000). Nature 403(6769):501-2

1-cell 6h 1-cell 8h 2-cell 22h 4-cell 45h

Adapted from Rougier et al. (1998). Genes Dev 12(14):2108 -13

4-cell asymmetrical staining 8-cell weak labeling, rare asymmetrical chr.

NOBODY EVER REPRODUCED THESE DATATHIS PAPER WAS NEVER RETRACTED

HW: What’s the closest mammalian homologue of ROS1?

Introduction and Background:What hypotheses are being tested in this paper?

What information induced the authors to perform the experiments?What new methods or insights brought to bear on the problem?

Methods:What are the critical methods of the paper?

What enabling technologies are used?What are the weaknesses of the methods used?

Are there other or better approaches that could be used?If this is a genetics approach, what would be a biochemical or molecular approach?

If this is a biochemical or molecular approach, what genetics methods could be used?

Results and DiscussionWhat are the primary conclusions of the paper?

Did the authors prove their hypotheses?What novel information or directions come from this work?

What control experiments were performed?What assumptions still remain in the work?How could these assumptions be tested?

What other explanations for the observations are still possible?

Reading the original literature ...

Journal Club ofAbysmal Disasters

• success stories of poor science• bad but Nature papers!• autopsies of crappy papers• examples one shouldn’t follow• retractions and bad reputations• learning from mistakes, which cost careers

… in English ...… once a month ...

… seminar room, first floor …… watch out of further announcements ...

The Institute of Molecular Genetics, PragueDepartment of Epigenetic Regulations

organizes:

Bad papers also deserve attention … therefore:

EPIGENETIKA MB150P85

Documents

Transcript of EPIGENETIKA MB150P85