6. cloning of genes of interest · 6.2 insertion mutagenesis - t-dna tagging 9 gene x wild type =...

6. Cloning of genes of interest6.1 Positional cloning6.2 Transposon cloning6 3 PCR l i b f h l6.3 PCR-cloning by use of homologue sequence6.4 Cloning of genes of interest in silico

7. Expressed Sequence Tags (ESTs) and EST-library7.1 RNA, mRNA and cDNA7.2 ESTs and EST/cDNA-libraries/databases7.3 SSH, Genechips and RNAseq technologies

GJ461DS467

6.1 Positional cloning – genetic and physical map 2

GJ17

GJ16

GJ162

GJ131

DS71GJ476

GJ461

GJ10GJ01

DS09

DS467

GJ367

GJ402

GJ403

GJ162 GJ10

GJ70GJ21

GJ18

GJ392 GJ511GJ367

GJ29

GJ44

GJ531

0.21.8 0.2 0.40.3 0.1 0.6 0.2 8.4SP6

T7

cM

3.8 cM

6.1 Positional cloning – Map Based Cloning 3

Ai l i f fPositional Cloning of the Hs1pro-1 Gens

• Aim: cloning of a gene of interest with an unknown gene product

1: Marker-IsolationHs1pro-1

• Method: Localisation of gene

1: Marker-Isolation

2: YAC/BAC-contig

Tl: 940043

• Method: Localisation of gene position by

1 genetic mapping 3: cDNA-Isolation1. genetic mapping2. physical mapping3. cloning of the interesting

i ( i )4: Genetic

Hs1pro-1

region (contig)4. Searching candidate

genes

complementation

6.1 Positional cloning – minimal tiling path 4

Hs1pro-1 A5-117-T7O6-100-T7 23a + 14b 128R 104RA906001

Pro4

A906001

A19-62 115kb

K15-57 120kbK15 57 120kb

O5-132 140kb

I15 137I15-137 145kb

G11-123 140kb

B.procumbens specific probe

i l

A23-67 130kb

T7-End Sp6-EndBAC-end-sequence

signal on BAC

6.2 Insertion mutagenesis - Transposon tagging 6

- Two elements: Ac / Ds:

Ac Activator1 2 copies per Cell1-2 copies per Cellaktive transposonconfers production of transposase

Ds Dissociation5-100 copies per Cell5 00 cop es pe Ceinaktive transposon (deleted Ac-element)no transposasecan be trans-activated by the Ac-element

6.2 Insertion mutagenesis - Transposon tagging 7

Phenotype Ie

(resistent) Cloning of the candidate geneg

e

Phenotyp II(susceptibel) no protein - MUTATION

mRNA disruption

Production of a mutant populationi f i t ti h t

7screening for interesting phenotypes

6.2 Insertion mutagenesis – inverse PCR 8

known sequence (e.g. transposon) UNKNOWN sequence

EcoRI EcoRI EcoRIEcoRI

Restriction

EcoRI EcoRI EcoRIEcoRI

PrimerdesignEcoRI EcoRIf

r

f

iPCR fragment

Ligationr

f

PCRelektrophoreses, cloning r

6.2 Insertion mutagenesis - T-DNA tagging 9

GENE X

Wild type = X+

T-DNAE XGENE X

Arbitrary primer (ap)

Nested specific primers1

23

PCR 1 with ap + 1(low stringency)

Mutant = x-

( g y)

PCR 2 with ap + 2

1 2 3

PCR 3 with ap + 3

6. Cloning of genes of interest6.1 Positional cloning6.2 Transposon cloning6 3 PCR l i b f h l6.3 PCR-cloning by use of homologue sequence6.4 Cloning of genes of interset in silico


6.3 PCR-cloning by use of homologue sequence 11

Amino acid sequences are highly conserved

X 21 (Ri X h )

Pto (Tomato, Pseudomonas )

Ser/Thr-Protein Kinase

N (Tabacco, TMV)

Xa21 (Rice, Xanthomonas)

Cf9 (Tomato, Cladosporium )

Cre3 (Wheat, Heterodera )

Prf (Tomato, Pseudomonas ) LZ: Leucine-ZipperNBS: Nucleotide-Binding SiteLRR: Leucin-rich RegionTMD: Trans Membrane Domaine

Gpa2 (Potato, Globodera)

Mi (Tomato, Meloidogyne )

TMD: Trans Membrane DomaineTIR: Toll/Interleukin-1 Signal domaine

Hero (Tomato, Globodera)

Gro1-4 (Potato, Globodera)

Hs1pro-1(sugar beet, Heterodera )

LRRLZ/CC NBS TMD


Resistance gene analogues (RGA) in Arabidopsis


R P-loop kinase-2 kinase-3a GLPL TIR/CC

Prf GLGKTTLAKKIY KRFLILIDDVW SNRSRIILTTR RGLPLSVVLV LKPCFLYFGGFL

Cre3 GSGKSTLAQFVY KRFLLVLDDVW KKGSKILVTTR KGSPLAARTV ARRCFAYCSIFP

Mi GSGKTTLAYKVY KRYLIVLDDVW KKGSRIILTTR KGLPLVADLI LKPCLLYFASFPMi GSGKTTLAYKVY KRYLIVLDDVW KKGSRIILTTR KGLPLVADLI LKPCLLYFASFP

Gpa2 GIGKTTLAAKLY RRYLVVIDDIW DNGSRILLTTR GGLPLAITLI LKPCFLYFAIFA

Hero GVGKTTLANKVY KRYLIVLDDVW ERGNRVILTSR KGLPLALVLI LKPLLLYFARLQ

Cons GSGKTTLA KRYLIVLDDVW KKGSRILLTTR KGLPLAAVLI LKPCFLYFAIFP

R-s1 R-as1

R-s1: G K T T L A

Design of degenerated primer

5´- GGN AAR ACN ACN YTN GC (deg.)

R-as1: K G L P L A5´- GC NAR NGG NAR NCC YTT (deg.)5 GC NAR NGG NAR NCC YTT (deg.)

H=A/T/C; R=A/G; N=G/A/T/C; s=sense; as=antisense

6.3 PCR with degenerated primer 15

Using the IUB-codes (International Union of Biochemistry)IUB Code

N (I) V B H D K S W M Y R

Basen A,C,G,T G,A,C G,T,C A,T,C G,A,T G,T G,C A, T A,C C,T A,G

Leucine Lysine Proline Cysteine Phenylalanine Leucine Tyrosine Alanine Isoleucine Phenylalanine Alanine

AA Leu Lys Pro Cys Phe Leu Tyr Ala Ile Phe Ala

Basen CAC A

AC C C

AC

AA C

AC

TT

GT

A AG

C C CGT

T GT

T TT T

T CGT

T AT

G C CGT

A TCT

T TT

G CGT

IUB Y T N A A R C C N T G Y T T Y Y T N T A Y G C N A T H T T Y G C N


Q L N A T L N HCAG CTG AAC GCG ACG CTG AAC CACCAA CTC AAT GCC ACC CTC AAT CAT

AS-Sequenz

CAA CTC AAT GCC ACC CTC AAT CATCTA GCA ACA CTACTT GCT ACT CTTTTG TTG

reverse TranslationTTG TTGTTA TTA

C G G G G3‘-GT AI TT CGI TGI AI TT GT-5‘

T A A A A Primer-SequenzT A A A A q

PCRPCR

Cloning and sequencing

H=A/T/C; R=A/G; I=G/A/T/C; s=sense; as=antisense

6.3 PCR amplification with degenerated primer 17

r s w r s w 4

Degenerated DegeneratedDegenerated Primer pair: R3/R6

DegeneratedPrimer pair: Gpa2s1/as1

6.3 PCR amplification with degenerated primer 18

Z-1

Mi, Prf, RPM1Z-3

Mi, Prf, Gpa2, Rx

Z-7

Mi, Prf, Gpa2, Rx

Mi, Prf, Gpa2, Rx

Z-9

Mi P f RPM1Mi, Prf, RPM1

6.4 Cloning of genes of interest in silico 20

Sequencing of largegenome regions g g

looking for ORFslooking for ORFs

Database searchAnnotation

? ?

Selection of did tcandidate genes

FunctionalFunctionalcharacterization

6. Cloning of genes of interes6.1 Positional cloning6.2 Transposon cloning6 3 PCR l i b f h l6.3 PCR-cloning by use of homologue sequence6.4 Cloning of genes of interst in silico


7.1 RNA, mRNA and cDNA 22

Genome

genomic DNA, gDNAR bi ti d t tiRecombination and mutation

Transkriptomep

mRNA, ESTs, cDNAEnvironment

Proteome

structural proteins, enzymesp , yEnvironment

Metabolome

Metabolites, secondary metabolitesEnvironment

7.1 RNA, mRNA and cDNA 23

cDNA-synthesis

5´ 3´AAAAAAA mRNA<TTTTTTTT 5´

i cDNAR primer cDNAfirst strand synthesis

ReverseTranscriptase (RT) = RNA dependent DNA-polymerase

Degradation of the RNA + <AAAAAAA 3´5´ second strand synthesis<TTTTTTTT 5´<AAAAAAA 3

3´5

Cloning (Plasmid vector)

Sequencing

257.2 EST/cDNA libraries

EST = Expressed Sequence Tags

Gene expression

depending on organs, developmental stage and environment

267.2 EST/cDNA libraries - EST assembling

ESTs = roughly sequenced transcripts (J. C. Venter)

tissue (e.g. roots)

RNA-preparation

cDNA synthesiscDNA synthesis

cDNA-clone library

random sequencing

high number of clones

t lgene catalog

7.2 EST/cDNA libraries 27

Construction of a cDNA-library

Phage library

odod.

7.2 EST/cDNA libraries 28

Construction of a cDNA-library

Plasmid-libraryAdaptor ligation

<GAGCTCTTTTTTTT 5´<CTCGAGAAAAAAA 3´

3´ CTTAAG5´ GAATTC

Adaptor ligation

EcoRI XhoI

EcoRI

Xh IXhoI

7.2 EST/cDNA libraries - screening 29

7.2 EST/cDNA libraries - screening 30

screen I screen II

317.2 EST/cDNA libraries

• ESTs are an evidence for transcription of a certain sequence

• ESTs are tools to identify new genes (dbEST-search)

• The relative amount of EST-sequences is proportional to its expression of a gene in one experiment > Possibility of a virtual northern

b t 60 i EST kabout 60 mio. EST-sequences are known

more than 10 mio. ESTs from 200 plant species, ordered by tissue, culture conditions etcculture conditions etc.

Species Family No. of ESTs

327.2 EST/cDNA libraries - EST assembling

7.2 EST/cDNA libraries - RACE 33

Region of known EST sequence

FROM EST TO VALID mRNA-SEQUENCE

Region of known EST-sequence

Region to be amplifiedby 5´-RACE

Region to be amplifiedby 3´-RACE

GSP2 NGSP2 NGSP1 GSP1Universalprimer

Universalprimer

NNAAAA-3NNTTTTT-5´_UPBS

UPBS_5´3´

RACE = Rapid Amplification of cDNA-Ends

7.3 SSH, Genechips and RNAseq technologies 35

Technologies of Transcriptome-analysis

Question: Which genes are involved in a certain trait?Question: Which genes are involved in a certain trait?

Possibility of identification of up- and down-regulated genes(i ti d ifi i t l diti / t t t )(in a tissue, under specific environmental conditions / treatments.....)

Expression profiling

Finding candidate genes for the trait (phenotype)

With sequence information

Withoutsequence informationq q

SSH ( )RNAseq

Genechips

7.3 Construction of an SSH-library 36

Resistant plants20 plants 20 plants 20 plants

3 DAI 6 DAI 12 DAI

Susceptible plants20 plants 20 plants 20 plants

3 DAI 6 DAI 12 DAI3 DAI 6 DAI 12 DAI 3 DAI 6 DAI 12 DAI

RNA-IsolationRNA RNA RNA RNA RNA RNA

pooling

RNA RNA RNA RNA RNA RNA

mRNA-RTester / DriverForward-SubtractionR-Gene

mRNA-S

Driver / TesterReverse-SubtractionForward Library Reverse Library

up-regulated genes down-regulated genes

Aim: Identification of genes related to R-gene mediated plant-Resistance

7.3 Principle of SSH-libraries 37

SSH- Subtraktive Supression Hybridisation

Adaptor-Ligation (F/R)

1. Hybridization

2. Hybridization

Amplification Cloning Sequencing

7.3 SSH library 38

No special equipment necessary (only sequencing instrument)☺ p q p y ( y q g )

S i bl f k d ( i i f )

☺

☺ Suitable for weak expressed genes (e.g. transcription factors)☺

Amplification of long cDNA-molecules (easy to annotate)☺

Analysis of known and unknown transcripts☺

One SSH-procedure for each comparison, time-point, tissue

7.3 Genechips (Microarrays) 39

1. Thousands of defined oligonukleotides are immobilised on an glass-array (chip)

2. Each oligonucleotide represents a transcript

3. Hybridisation with fluorescence-labeled cDNA from different tissues, environments, treatmrents...

Oligonucleotides as probes at a

labeled cDNA-molecules

Hybridisation with the complementary oligonucleotideas probes at a

specific positionon the array

oligonucleotide

7.3 Genechips (Microarrays) – differential hybridization 40


upregulated in treatment A

upregulated in treatment B(= downregulated in treatment A)

no expression in A and B

equal expression in A and B

7.3 Genechips (Microarrays) 43

Special equipment necessary (expensive)

Only known genes can be analysed

Weak and strong expressed genes can be analysed☺

> 100 Arrays per production process☺high number of quick and easy experimentshigh number of quick and easy experimentsonly RNA-isolation, labeling and hybridization

7.3 RNAseq – trancriptome sequencing 44

New generation sequencing of cDNA libraries(454 Illumina )(454, Illumina....)

Quantitative analysis of gene expression Q y g pby counting reads per transcript

7.3 RNAseq – trancriptome sequencing 45

Sanger Roche454

IlluminaHiSeq2000g 454 HiSeq2000

Read length: 1000 bp 400 bp 100 bp

Sequenz-informationper run

0,0003 GBp 0,6 GBp > 200 GBpper run


After performing an expression profiling experiment

1 I ili l i l ti f i t ti did t1. In silico analysis selection of interesting candidate-genesfrom a huge ammount of data

2. Verification of expression profile results by an independentexperimental system

3. Functional characterization of candidate genes


1. In silico analysis selection of interesting candidate-genesfrom a huge ammount of data

1000

TreatmentsA B C D

Trea

tmen

t A

10

100

1 10 100 1000

1

Genetic Characteristic WT (raw)

Treatment B

Cell rescue, defense,cell death and ageing

16% Cellular communication/

Cell growth, celldivision and DNA synthesis

2%16% Cellular communication/

signal transduction

5%

Cellular organization2%

Cellular transport andtransport mechanisms

Unknown34%

6

p4%

Energy4%Transcription

5%

Protein-Synthese14%

Protein-Destination4%

Metabolism10%


2. Verification of expression profile results by an independentexperimental system

qPCR N th

Gene 1

qPCR Northern

Gene 2

Gene 1

Gene 1 Gene 2 PR2 Gen 5 Gen 6Gene 3 Gen 7Gene 4 HK

Gene 4

Gene 3

Gene 1 Gene 2 PR2 Gen 5 Gen 6Gene 3 Gen 7Gene 4 HK


3. Functional characterization of candidate genes

1. Genetic complementation

2. Knock out mutants / RNAi2. Knock out mutants / RNAi

3. Protein function / protein assays

4. Promoter analysis

G HG H

Thank you for your attention.

6. cloning of genes of interest · 6.2 insertion mutagenesis - t-dna tagging 9 gene x wild type =...

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