Flanking Sequence Tags identified by 3’-RACE

Laurence Meslet-Cladière and Olivier Vallon

UMR 7141 CNRS/UMPC,

Institut de Biologie Physico-Chimique

13 rue Pierre et Marie Curie, 75005 Paris, FranceTel : +33 1 5841 5058 Fax : +33 1 5841 5022 E-mail : ovallon @ibpc.fr

Summary

• Chlamydomonas reinhardtii is a well-established model organisms to study photosynthesis, chloroplast biology etc...... and to develop biodiesel technologies

• Random insertional mutagenesis is a powerful way to study gene function, incl. at the genome scale. But once a library has been established, it is not trivial to identify the locus where insertion occurred. PCR-based methods to identify Flanking Sequence Tags (TAIL-, RESDA-, GenomeWalker-, SiteFinder-PCR etc) are sometimes difficult in the 65% GC DNA of Chlamydomonas. We have sought to develop a method for FST determination that does not require the annealing of degenerate oligos.

• Using a novel Spectinomycin resistance marker (recoded version of the bacterial AadA gene), we show that a high number of transformants can be obtained with a truncated version of the cassette, lacking a 3’-UTR. Antibiotics resistance depends upon the expression of a chimeric RNA, whose 3’-UTR is provided by the flanking Chlamydomonas DNA.

• We have used classic 3’-RACE to amplify the chimeric AadA transcript from randomly-selected transformants. Out of ... strains analyzed, we were able to extract FST from ... strains, and ... of them could be uniquely mapped to the genome.

• We propose this methodology as an alternative to DNA-based PCR methods for the large scale sequencing of insertional mutant libraries.

A recoded AadA marker

RBCS2 chloroplast Transit Peptide

AtpA N-term

acgt positions

modifiedmanually

BstAPIBsgI

MunI

NdeIBbvCI

BmtINheISmaIXmaIAscI

SphI

MauBIAatIISfiI

MunIFspAIFspI

PpuMI

PfoI

AatIIBamHI

BclIXcmI

Acc65IKpnI

FspI

PsiI

XmnI

ScaI

FspI

NmeAIII

PciI

SapI

AleISacII

NotI

HSP70A-Pro

RBCS2-Pro5' UTR

CrAadA

RBCS2-3'polyAf1 (+) ori

bla

pUC ori

pALM32

pALM32 : AR_CrAadA_3’-RBCS2

A

BCD

A, B, C, D: primers for PCR

0

100

200

300

400

500

600

700

800

900

vecteur-

AR-

AadA-3'

AR-

AadA-3'

R-AadA-

3'

AadA-3' AR-

AadA

AadA

co

lon

ies

/ t

ran

sfo

Spec 75 µg/ml

Spec 100 µg/ml

Spec 200 µg/ml

Transformation by cut plasmid

The CrAadA marker can transform even when deprived of promotor or 3’ UTR

Removal of the 3’-UTR only marginally reduces transformation rate

Transformation by PCR products

blu

nt

5'

uncut

blu

nt

5'

uncut

blu

nt

5'

uncut

0

200

400

600

800

1000

1200

1400

1600

1800

2000

Tra

nsfo

rmation e

ffic

iency (norm

.)75 µg/ml

200 µg/ml

AR_CrAadA AR_CrAadA_3 ’UTR AR_CrAadA_3 ’UTR_T7pro

Generating FSTs by 3’-RACE

Marker DNA FST

AAAAAAmRNA 5’ 3 ’

TTTT primerA_B

Reverse transcription

1rst strand cDNA 3’ 5 ’TTTT TTTT primerA_B

1rst amplification

TTTT TTTT primerA_B

GSP1 B

SEQUENCING

2nd, nested, amplification

TTTT TTTT primerA

GSP2 A

3’-RACE method: Scotto-Lavino et al, Nat Protoc. 2006;1:2742-5

C1 C2 #2.2 #2.4 #2.6 #3.1 #3.3 #4.1 #4.2 #4.3 #14.2 #14.3 C1 C2 #2.2 #2.4 #2.6

CrAadA control: endogenous PETC

10 randomly picked strains, reverse-transcribed with primer QT

PC

R 1

PC

R2

*

* **

*

*

*

**

*

**

* ***

*

****

* : bands that gave a FST

#2.1 #2.3 #2.5 #3.2 #3.4 #3.5 #3.6 #4.4 #4.5 #4.6 #14.1 #14.4 C1 C2 C3 C1 C2 C3

Sequencing of whole PCR reaction

PCR2 PCR 1

>#4.4_CrAadA_F4 -- unclipped

GGGRTKAGCAGTATCTAGACGTCRACCCACTCTAKAGGATCCCCGCTCCGTGTWAATGGAGGCGTACGTASACACTGGGGGAGACTTTCCGTCACGGCCCCRWCCCARCGTC

GCTGGCAACGTCCACAGMTGTGCRCACCACGCGGCGCTGCTCACTCGCTGCCRACACGACSGCTCCCCGGCCCTGCCGCGGMCMTGCAGGTGRTCAAGGTGTTTGTAAGCKT

ATACAGTGACRACTACGGCAAGCGAGTGGCCATGGAGAACCTGCAGCGCCTGGAGCCSTRAGTGTCCRCRGGSRCCGGGGGGCATSKGACGAKGCATCKKGGCGGGGATGGA

AARGYSRGGGATGRCATCCSGGWGCRGGAGGGGTCSAGGAKTGAGGTGSGGCTGCGGGCCCACTTGRWGGMTAGTCTGTGSCMGCMGMTTGRCGTTTTCAGGGCCGCCACGG

CGCGTGTGACGGTCGASGACAGCGGACTCTCGCCACATCACACCGCRATCTGCTGCAGCTCACATGTAACCGTACCATACACRAAAAAAAAAAAAAAAAAARRWKKKKSKCY

KYYYCACAYAAAACGARKACTTTGAGGACCCARACGAGSGGCCGCAGGAGTACCCCAACCCCTTTGGCGACCTGTTMAWMRAMRACMRCGAKTACCGGRC

ARTGGCGGKCAAKCGCGTGSAGGAGCGGASGCGTAGCCAGGGCCKCCCGCASCCRCAAGGYCGCGGGCAKGGGCKGCRTGTGYAAKGGCAGCAGCGGGAGGMKGCGGCGGCT

GAGACTGGTGATGASTAGGTATAATGTCTGTTTGTCAGTGTATACTAACGAGCACGTGCGGGYRCGTGCAGGAACRGTGGATSGKCYGCWGYATGCAGGTTCATTGATAKCG

CAGTGCSACSGGAGCACGGRGCCTCMGGCACGCAAGAGCTACTKGWCCTACTGCTAGAGTMCGTCCTMCGGYGCTAGTCAGATCSCRCCTGGGAATCATCTTCTYGCCTKGT

GCGATGGWACGRGGTAAGGGGCAAGGATGCYGWATYCTGGMATCRYCTCSMCCGGTGCATCTTCYCCCCATTWACGTARCAGTTKCAYKSKTG

Au9.Cre02.g145000 Ribosome-binding factor A

NB: several sites of polyadenylation ; an intron is spliced out in the newly generated 3’UTR

24 « difficult » strains, reverse-transcribed with another primer, Qs

>QS

CGAGATCTACACTCTTTCCCTACACTAGACGACGCTCTTCCGATCTTTTTTTTTTTTTTTTTTT

>QU Tm=63.2°C

CGAGATCTACACTCTTTCCCTACACT

>QD Tm=64.4°C

CACTAGACGACGCTCTTCCGATCT

PCR1#2.6 2.11 3.4 3.7 3.8 3.12 3.11 4.3 4.5 4.6 4.9 4.12

11.3 11.5 11.6 11.8 11.9 11.10 11.11 11.12 14.2 14.3 14.4 con

PCR2#2.6 2.11 3.4 3.7 3.8 3.12 3.11 4.3 4.5 4.6 4.9 4.12

11.3 11.5 11.6 11.8 11.9 11.10 11.11 11.12 14.2 14.3 14.4 con

Summary of FSTsstrain

cassette

end

end

modified longest FST on genome gene annotation

position in

gene

cassette

/gene

polyadenylation

signal

#2.1 uncut -9 chromosome_16:304054-303727 Cre16.g649800PPR3;

Pentatrichopeptide

intergenic

(donwstream) + not reached

#2.2 uncut 0chromosome_1:8429714-8429732, spliced to

following exonsCre01.g060850

PSBS3, Chloroplast

Photosystem II- 3rd exon + not reached

#2.3 uncut 0 chromosome_12:589974-589850 Cre12.g488050FFT5, Fructan

fructosyltransferase intron 2 - not reached

#2.4 uncut 0 chromosome_14:3326333-3326254 Cre14.g630200 no predicted function3' UTR +

TGTAA (-17), that of

host gene

#2.5 uncut 0 chromosome_12:3269654-3269776 Cre12.g512250HRDC domain,

POLYMYOSITIS/SCLE 4th exon + TGTTC ? (-37)

#2.6 uncut 0 many locationsTEs: Toc1 and

DNA-2-7_CRnot reached

#2.7 uncut ?chromosome_10:1630531-1630475 spliced to

1630157-1630059Cre10.g429850 no predicted function

7th exon + not recognizeable

#2.8 uncut ? scaffold_27:75383-75179 Cre27.g774700SGNH hydrolase

(GDSL hydrolase); 5'UTR + not recognizeable

#2.9 uncut -3chromosome_9:2341565-2341083

NB: same as #4.3 !Cre09.g400950

Pfam:07690 Major

Facilitator Superfamily

3'UTR

(endogeneous) + TGTAA (-20)

#2.11 uncut 0 chromomsome_17:1621018-1620917 Cre17.g708000 PAS domain proteinvery end of the

3'-UTR + TGTGA (-33)

#3.1 BsiWI +4chromosome_3:1474847-1474820... and

chromosome_3:1475170-1475092…Cre03.g159300

SMALL MEMBRANE

PROTEIN-RELATEDnot reached

#3.2 BsiWI +3 chromosome_12:9166015-9166885 Cre12.g560350CNK2; NimA-related

protein kinase 2 intron 1 + TGTAA (-19)

#3.3 BsiWI +4 chromosome_7:1015855-1015936 Cre07.g319550conserved hypothetical

protein with FIST_C intron 1 +

TGTCC (-6) or

TGTCC(-11)/ TGGAA

#3.5 BsiWI -5a sequence similar to chromosome_7:2564723-

2565098 and other locations

exact location

unknown, similar to

unknown function (a

sequence repeated >12 ? ? TGTAA (-18)

#3.6 BsiWI +1 chromosome_3:2440121-2440156 Cre03.g166950PGM5;

phosphoglycerate intron 6 + TGTAA (-7)

#3.7 BsiWI ? chromosome_14:3746617-3744745 Cre14.g632700 protein kinaseintron 19 on

Au10.2 model + not reached

#3.11 BsiWI +4chromosome_17:2083314-2083570 with

junction, but also chromosome_17:2083567-Cre17.g712100 MDAR1

intron 7 + TGTGA (-4)

#4.1 (SnaBI) 0 chromosome_5:377208-377154 and -377020 Cre05.g231500 Zn-finger protein intron 6 + not recognizeable

#4.2 (SnaBI) 0not in v4; in finishing draft:

8022_2:24991-25170? ? ? ? Not reached

#4.3 SnaBI 0Chromosome_9:2341490-2341445

NB: same as #2.9 !Cre09.g400950

membrane protein of

Major facilitator 3' UTR + not reached

#4.4 (SnaBI) 0 chromosome_2:9598215-9597761 Cre02.g115000Ribosome-binding

factor Aintron 4 +

TGTAA (-18) / not

reached

#4.8 (SnaBI) chromosome_7:698506-698252 Intergenic TGGTAC ? (-40)

#4.10 (SnaBI) ? chromosome_7:4591244-4590811 Cre07.g346000 unknown function end of 3'UTR + not recognizeable

#11.1 AatII ? chromosome_16:2391524-2391584 Cre16.g666300 protein kinaseupstream,

integernic+ TGTAA (-19)

#11.4 AatII 2 chromosome_4:704462-705281 Cre04.g215800 no annotation last exon + not reached

#14.1 uncut ? chromosome 14:2909515-2909394 Cre14.g627600 Dynein heavy chainintron 6 and

exon 8+ not reached

#14.2 uncut 0

ambiguous: chromosome_12:4869726-

4870332 or 4925725-4926331 or 4944276-

4944882 (all with 1 intron)

Cre12.g526150 or

Cre12.g526450 or

Cre12.g526650

Protein kinases intron 3 + TGTGA (-4)

Total tested: 35 Failures: 8 Mappable FSTs: 27 Genome verified (thus far): 6

• PCR product undigested or digested with SnaBI or BsiWI

GGCGTACGTAGACAC-3’CCGCATGCATCTGTG-5’

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End-resection

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