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TABLE 1 Oligonucleotide primers and probes used in dot blots and DGGE analysis

TABLE 1 Oligonucleotide primers and probes used in dot blots and DGGE analysis. PCR Primers. Specific target DNA. Sequence 5´-3´. OXY107F*. Oxygenic phototroph biased. GGA CGG GTG AGT AAC GC G TG R. OXY1313R*. Oxygenic phototroph biased. CTT CAC GTA GGC GAG TTG CAG C. CYA359F†.

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TABLE 1 Oligonucleotide primers and probes used in dot blots and DGGE analysis

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  1. TABLE 1 Oligonucleotide primers and probes used in dot blots and DGGE analysis PCR Primers Specific target DNA Sequence 5´-3´ OXY107F* Oxygenic phototroph biased GGA CGG GTG AGT AAC GC G TG R OXY1313R* Oxygenic phototroph biased CTT CAC GTA GGC GAG TTG CAG C CYA359F† Oxygenic phototroph specific GGG GAA TYT TCC GCA ATG GG PRO1017R Prochlorococcus, excluding MIT9303, MIT9313, TCC CGA AGG CAC CCT CWA AA MIT9302 27F Eubacterial AGA GTT TGA TCM TGG CTC AG 1224R Eubacterial CAT TGT AGC ACG TGT GTA Oligonucleotide Probes Specific target DNA Sequence 5´-3´ S1PRO634R HLI Prochlorococcus GCC GAT CAG TTT CCA CTG S2PRO634R HLII Prochlorococcus GCC TTT CAG TTT CCA CTG DPRO634R LL Prochlorococcus (except SS120) GCC AAT CAG TTT CCA CTG SARG634R SS120 GCC CTT CAG TTT CCA CTG MIT1023R MIT9303 TGC GTT CCC AAA GGC ACT EUB338 Eubacteria GCT GCC TCC CGT AGG AGT PRO444R ‡ Prochlorococcus , excluding MIT9303, MIT9313 TAT TCC TCA AGT ACC GTC ATA MAR572R Marine picophytoplankton clade biased GCC GCC TGC GGA CGC TTT . A GC-clamp consisting of a 40 nucleotide GC-rich sequence (5´-CGC CCG CCG CGC CCC GCG CCG GTC † CCG CCG CCC CCG CCC-3´) was attached to the 5´ end of the primer and used in DGGE analysis . ‡ Based on SAR6R .

  2. TABLE 2 Oligonucleotide primers and probes used in FISH* Oligonucleotide Probes Specific target DNA Sequence 5´-3´ S1PRO634R HLI Prochlorococcus GCC GATCAG TTT CCA CTG 645 HLI* HLI Prochlorococcus ACC ATA CTC AAGCCG ATC S2PRO634R HLII Prochlorococcus GCC TTTCAG TTT CCA CTG 645 HLII* HLII Prochlorococcus ACC ATA CTC AAGCCT TTC DPRO634R LL Prochlorococcus (PAC1/NATL1-MIT cluster) GCC AATCAG TTT CCA CTG 645 LL* LL Prochlorococcus (PAC1/NATL1-MIT cluster) ACC ATA CTC AAGCCA ATC 181 HLI* HLI Prochlorococcus CAT TTC ACC TAT CGG CAT . 181 LL* LL Prochlorococcus (not MIT9211 & includes some Syns) CAT TTC ACC TCT CGG CAT 148 LL-MIT* LL Prochlorococcus (MIT9211,MIT9303, MIT9313 only) CCG TTT CCA ACC GTT ATC 405PRO* Gen Prochlorococcus (General, but notMIT9303, MIT9313) AGA GGC CTT CGT CCC TCA

  3. Prochlorococcus sp. MIT9107 Prochlorococcus sp. SB Prochlorococcus sp. TAK9803-2 Prochlorococcus sp. MIT9215 645HLII Prochlorococcus sp. MIT9202 Prochlorococcus sp. MIT9201 Prochlorococcus sp. MIT9302 Prochlorococcus sp. GP2 405PRO Prochlorococcus sp. MIT9312 Prochlorococcus sp. EQPAC1 645HLI 181HLI Prochlorococcus sp. PCC9511 Prochlorococcus sp. CCMP1378 Prochlorococcus sp. NATL2A 645LL 1445NATL Prochlorococcus sp. NATL2B 181LL Prochlorococcus sp. PAC1 Prochlorococcus sp. NATL1MIT Prochlorococcus marinus SS120 Prochlorococcus sp. MIT9211 Prochlorococcus sp. MIT9303 Prochlorococcus sp. MIT9313 405SYN 181LL Synechococcus sp. WH8018 Synechococcus sp. WH7805 Synechococcus sp. WH7803 Synechococcus sp. WH8103 Synechococcus sp. WH8101 181LL Synechococcus sp. PCC6307 Synechococcus sp. PCC6301 0.1

  4. TABLE 1 Oligonucleotide primers and probes used in dot blots and DGGE analysis PCR Primers Specific target DNA Sequence 5´-3´ OXY107F* Oxygenic phototroph biased GGA CGG GTG AGT AAC GC GTG R OXY1313R* Oxygenic phototroph biased CTT CAC GTA GGC GAG TTG CAG C CYA359F† Oxygenic phototroph specific GGG GAA TYT TCC GCA ATG GG PRO1017R Prochlorococcus, excluding MIT9303, MIT9313, TCC CGA AGG CAC CCT CWA AA MIT9302 27F Eubacterial AGA GTT TGA TCM TGG CTC AG 1224R Eubacterial CAT TGT AGC ACG TGT GTA Oligonucleotide Probes Specific target DNA Sequence 5´-3´ S1PRO634R HLI Prochlorococcus GCC GAT CAG TTT CCA CTG S2PRO634R HLII Prochlorococcus GCC TTT CAG TTT CCA CTG DPRO634R LL Prochlorococcus (except SS120) GCC AAT CAG TTT CCA CTG SARG634R SS120 GCC CTT CAG TTT CCA CTG MIT1023R MIT9303 TGC GTT CCC AAA GGC ACT EUB338 Eubacteria GCT GCC TCC CGT AGG AGT PRO444R ‡ Prochlorococcus , excluding MIT9303, MIT9313 TAT TCC TCA AGT ACC GTC ATA MAR572R Marine picophytoplankton clade biased GCC GCC TGC GGA CGC TTT A GC-clamp consisting of a 40 nucleotide GC-rich sequence (5´-CGC CCG CCG CGC CCC GCG CCG GTC † CCG CCG CCC CCG CCC-3´) was attached to the 5´ end of the primer and used in DGGE analysis . ‡ Based on SAR6R

  5. S1PRO634R HLI Prochlorococcus GCC GATCAG TTT CCA CTG 645 HLI* HLI Prochlorococcus ACC ATA CTC AAGCCG ATC S2PRO634R HLII Prochlorococcus GCC TTTCAG TTT CCA CTG 645 HLII* HLII Prochlorococcus ACC ATA CTC AAGCCT TTC 645 LL* LL Prochlorococcus (PAC1/NATL1-MIT cluster) ACC ATA CTC AAGCCA ATC 181 HLI* HLI Prochlorococcus CAT TTC ACC TAT CGG CAT 181 LL* LL Prochlorococcus (not MIT9211 & includes some Syns) CAT TTC ACC TCT CGG CAT 148 LL-MIT* LL Prochlorococcus (MIT9211,MIT9303, MIT9313 only) CCG TTT CCA ACC GTT ATC 405PRO* Gen Prochlorococcus (General, but not MIT9303, MIT9313) AGA GGC CTT CGT CCC TCA TABLE 2 Oligonucleotide primers and probes used in FISH* Oligonucleotide Probes Specific target DNA Sequence 5´-3´ DPRO634R LL Prochlorococcus (PAC1/NATL1-MIT cluster) GCC AATCAG TTT CCA CTG .

  6. Prochlorococcus sp. MIT9107 Prochlorococcus sp. SB Prochlorococcus sp. TAK9803-2 Prochlorococcus sp. MIT9215 645HLII Prochlorococcus sp. MIT9202 Prochlorococcus sp. MIT9201 Prochlorococcus sp. MIT9302 Prochlorococcus sp. GP2 405PRO Prochlorococcus sp. MIT9312 Prochlorococcus sp. EQPAC1 645HLI 181HLI Prochlorococcus sp. PCC9511 Prochlorococcus sp. CCMP1378 Prochlorococcus sp. NATL2A 645LL 1445NATL Prochlorococcus sp. NATL2B 181LL Prochlorococcus sp. PAC1 Prochlorococcus sp. NATL1MIT Prochlorococcus marinus SS120 Prochlorococcus sp. MIT9211 Prochlorococcus sp. MIT9303 Prochlorococcus sp. MIT9313 405SYN 181LL Synechococcus sp. WH8018 Synechococcus sp. WH7805 Synechococcus sp. WH7803 Synechococcus sp. WH8103 Synechococcus sp. WH8101 181LL Synechococcus sp. PCC6307 Synechococcus sp. PCC6301 0.1

  7. HLI LL HLI LL HLIILL HLII LL LL Distribution of Prochlorococcus genotypes • Eastern North Atlantic • stratified HLI and LL genotypes correlated with mixed layer depth  in agreement with clone libraries, DGGE analysis and FISH data • Western North Atlantic • uniform distribution of HLII and LL genotypes in a mixed water column • Mediterranean Sea • stratified HLI and LL genotypes as observed in the eastern North Atlantic • Red Sea • stratified HLII and LL genotypes

  8. Prochlorococcus dot blot data : Coastal Chile (20.4°S, 70.6°W) low oxygen environment Location/Depth (m) Relative hybridisation (%) HLI LL HLII SARG AM2 15 m (20.4°S, 70.6°W)* 0.2 14.3 0.5 1.8 AM1 55 m (20.4°S, 70.6°W)** 0.2 40.8 1.1 0. 6 * above the oxycline ** below the oxycline 15m 1.7 5.4 0. 8 7.1 40m 2.1 40.1 1.4 1.3 50m 0.4 26.8 0.9 0.8 60m 0.4 65.2 1.7 0.7 40m - - - - 50m 1.4 26.7 1.0 0.9 60m 3.4 126.9(?) 2.7 5.1 70m 0.6 50.8 1.3 0.9

  9. Indian Ocean - Arabian Sea - cruise Sept 2001 • oligotophy/eutrophy gradient of surface waters along the transect • oxygen-depleted (ca 2-10 µM O2) waters that occur at depths from ca 200 - 2500 metres • throughout the basin. • divergent driven, gentle but continual upwelling of the Equator and adjacent downwelling waters

  10. Marine Synechococcus strains: Roscoff Strain Origin Isolator Location PUB:PEB F. Partensky PEB>PUB Mediterranean Sea 39°10’N 6 °10’E Minos 1 39°10’N 6 °10’E PEB>PUB Mediterranean Sea F. Partensky Minos 2 Minos 11 Mediterranean Sea PEB>PUB 34°0’N 18 °10’E D. Vaulot PEB>PUB 34°0’N 18 °10’E Minos 12 Mediterranean Sea D. Vaulot Max 42 D. Vaulot 26°18’N 63°26’W PEB<PUB Sargasso Sea Almo 3 F. Partensky Alboran Sea 36°11’N 1°51’W PEB<PUB Eum 14 F. Partensky Tropical Atlantic 21°02’N 31°0’W PEB<PUB PEB<PUB D. Vaulot Equatorial Pacific 5°N 150° 0°W Oli 31

  11. Strain Date of Depth of Colour PUB/PEB Motility N source ntcA RFLP 16S sequence isolation isolation ratio type RS9901 29.3.99 0 m Red 0.7 - NO3-/NH4+ I + RS9902 29.3.99 0 m Orange 1.4 - NO3-/NH4+ II + RS9903 11.5.99 10 m Orange 2.0 - NO3-/NH4+ III + RS9904 14.6.99 10 m Orange 1.5 - NO3-/NH4+ III + RS9905 18.7.99 10 m Orange/Red 1.0 - (+) NO3-/NH4+ IV + RS9906 23.8.99 10 m Green - - NO3-/NH4+ N/A + RS9907 23.8.99 10 m Red 0.5 - NO3-/NH4+ II + RS9908 7.9.99 10 m Red 0.5 - NO3-/NH4+ II + RS9909 7.9.99 10 m Green - - NO3-/NH4+ N/A + RS9910 7.9.99 10 m Red 0.5 - NO3-/NH4+ II + RS9911 11.5.99 10 m Red 0.8 - NH4+ V + RS9912 23.8.99 10 m Red 0.5 - NH4+ N/A + RS9913 7.9.99 10 m Green - - NH4+ N/A + RS9914 18.10.99 10 m Green - - NH4+ N/A + RS9915 18.10.99 10 m Red 0.8 - NH4+ IV + RS9916 22.11.99 10 m Red 0.7 - NH4+ I + RS9917 22.11.99 10 m Green - - NH4+ N/A + RS9918 22.11.99 10 m Green - - NH4+ N/A + RS9919 22.11.99 50 m Orange NO3- V - RS9920 22.11.99 150 m Orange NO3- VI - Analysis of Synechococcus strains III : Red Sea

  12. Synechococcus genes being used for multi-locus sequence typing and community structure analysis GeneEncoding rpoC1 DNA-dependent polymerase petB/D fragments b-type cytochrome & subunit of the photosynthetic & intergenic region cytochrome b6/f complex, respectively rnpB ribonucleoprotein enzyme (RNase P) pstS periplasmic phosphate-binding protein 16S rDNA small subunit rRNA ntcA global N transcriptional regulator

  13. rpoC1 pstS Distinguishing alleles petBD intergenic region - sequence length rpoC1pstS 5% divergence 16S rDNA 1% divergence petB-D 16S rRNA

  14. 14C labelling of DNA : growth of Prochlorococcus NATL1-MIT on 14C NaHCO3 12C 12C 14C 14C 55,000 x g o/n 40,000 x g o/n

  15. Environmental DNA received from partners : ROSCOFF BLANES HELGOLAND Name DNA amplifiable RA000404 x x RA000609 faint  RA000709 faint x RA001006   Name DNA amplifiable BL000921   BL001221   Name DNA amplifiable HE000803   HE001005   HE001206 x x Full length clone libraries generated : pA (8-28) pHr (1542-1522) Site Primers Identifier HE000803 pA/pHr He(H) 25/02/01 Mio (Prosope cruise) pA/pHr Mio(M) 25/02/01 Dyf (Prosope cruise) pA/pHr Dyf (D) 25/02/01

  16. Prosope cruise track

  17. Analysis of full length environmental clone libraries Sample Name DYF MIO Helgoland Number of clones 70 70 75 % Containing Insert 95.7 98.6 98.6 % Amplified with Primer couple 359F/1313R 72.9 72.9 61.3 (oxygenic-phototroph specific) % Amplified with Primer couple 359F/1038R 14.3 22.9 9.3 (1038r amplifies all marine Syns & Pros, ACE/PS680 cluster, PCC9005 = marine cyano-specific)

  18. Sequence Closest BLAST match identity DYF13 Prochlorococcus ENATL4, PAC1 99% DYF33 Prochlorococcus ENATL4, PAC1 99% HE24 env.SAR7; Synechococcus MIO12 Prochlorococcus MIT9211 99% MIO31 Prochlorococcus ENATL4, PAC1 99% MIO43 Prochlorococcus ENATL4, PAC1 99% MIO60 Prochlorococcus MIT9211; MIT9313 99% DYF18 env. Plastid seqs. OCS20; OM283 (cryptomonad) 99% DYF25 env. Plastid seqs. OCS50, OM153; (Haptophyte) 97% DYF38 Skeletonemapseudocostatum, uncult. Bacillariophyte 96% env. Plastid seq. Diatom DYF45 Chrysochromulinapolyepsis, env. Plastid OCS31; 98% HE3 HE3 Chrysochromulinapolyepsis, env. Plastid OCS31; 98% DYF45 HE12 env. Seq. OCS50; E. huxleyi plastid 100% HE17 env. Plastid seq. OM81; Ochromonas sp. plastid 93% MIO26 env. Plastid OCS31, OM21; 98% Sequence analysis of Cyanobacterial/plastid sequences from full length clone libraries

  19. Analysis of 107F-1038R environmental clone libraries Site Total no. of clones No. of unique RFLP types No. of unique clones Coverage (%) (HaeIII + EcoRI) HE000803 67 8 5 92.5 HE001005 100 34 24 76.0 RA000609 80 20 11 86.25 RA001006 72 13 7 90.3 BL000921* 82 13 8 90.2 BL001221 82 16 9 89.0 * of 9 clones partially sequenced 8 are Syns or Pros 1 has closest homology to a magnetitie containing magnetic vibrio but the identity is low (85.2%)

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