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Identification of proteins involved in the functioning of Riftia pachyptila symbiosis by Subtractive Suppression Hybridization.

Sanchez S, Hourdez S, Lallier FH - BMC Genomics (2007)

Bottom Line: Overall, half of the contigs matched records found in the databases with good E-values.Quantitative PCR analyses were congruent with our libraries results thereby confirming the existence of tissue-specific transcripts identified by SSH.Some of the keys to understanding metabolite exchanges may remain in the sequences we could not identify (hypothetical proteins and no similarity found).

View Article: PubMed Central - HTML - PubMed

Affiliation: Equipe Ecophysiologie: Adaptation et Evolution Moléculaires, UMR 7144 CNRS UPMC, Station Biologique, Place Georges Teissier, BP 74, 29682 Roscoff Cedex, France. sanchez@sb-roscoff.fr

ABSTRACT

Background: Since its discovery around deep sea hydrothermal vents of the Galapagos Rift about 30 years ago, the chemoautotrophic symbiosis between the vestimentiferan tubeworm Riftia pachyptila and its symbiotic sulfide-oxidizing gamma-proteobacteria has been extensively studied. However, studies on the tubeworm host were essentially targeted, biochemical approaches. We decided to use a global molecular approach to identify new proteins involved in metabolite exchanges and assimilation by the host. We used a Subtractive Suppression Hybridization approach (SSH) in an unusual way, by comparing pairs of tissues from a single individual. We chose to identify the sequences preferentially expressed in the branchial plume tissue (the only organ in contact with the sea water) and in the trophosome (the organ housing the symbiotic bacteria) using the body wall as a reference tissue because it is supposedly not involved in metabolite exchanges in this species.

Results: We produced four cDNA libraries: i) body wall-subtracted branchial plume library (BR-BW), ii) and its reverse library, branchial plume-subtracted body wall library (BW-BR), iii) body wall-subtracted trophosome library (TR-BW), iv) and its reverse library, trophosome-subtracted body wall library (BW-TR). For each library, we sequenced about 200 clones resulting in 45 different sequences on average in each library (58 and 59 cDNAs for BR-BW and TR-BW libraries respectively). Overall, half of the contigs matched records found in the databases with good E-values. After quantitative PCR analysis, it resulted that 16S, Major Vault Protein, carbonic anhydrase (RpCAbr), cathepsin and chitinase precursor transcripts were highly represented in the branchial plume tissue compared to the trophosome and the body wall tissues, whereas carbonic anhydrase (RpCAtr), myohemerythrin, a putative T-Cell receptor and one non identified transcript were highly specific of the trophosome tissue.

Conclusion: Quantitative PCR analyses were congruent with our libraries results thereby confirming the existence of tissue-specific transcripts identified by SSH. We focused our study on the transcripts we identified as the most interesting ones based on the BLAST results. Some of the keys to understanding metabolite exchanges may remain in the sequences we could not identify (hypothetical proteins and no similarity found). These sequences will have to be better studied by a longer -or complete- sequencing to check their identity, and then by verifying the expression level of the transcripts in different parts of the worm.

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Typical PCR profiles obtained after amplification of fragments of interesting cDNAs. S = subtracted sample; UN = unsubtracted sample. (A) and (B) Abundant tissue-specific transcripts: exosqueleton β-chitin-binding transcript (A) and galaxin transcript (B). (C) and (D) Transcripts enriched after SSH procedure: chitinase precursor transcript (C) and RpCAtr (D). (E) and (F) Rare transcripts enriched after SSH procedure: RpCAbr transcript (E) and intracellular globin transcript (F). (G) Abundant transcript in one tissue and rare in other tissue: MVP transcript. (H) Non equally subtracted transcript: cytochrome c oxidase subunit I transcript. The faint bands appearing at a smaller size than expected in some wells are interpreted as non-specific amplification (possible primer dimerization under specific conditions).
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Figure 2: Typical PCR profiles obtained after amplification of fragments of interesting cDNAs. S = subtracted sample; UN = unsubtracted sample. (A) and (B) Abundant tissue-specific transcripts: exosqueleton β-chitin-binding transcript (A) and galaxin transcript (B). (C) and (D) Transcripts enriched after SSH procedure: chitinase precursor transcript (C) and RpCAtr (D). (E) and (F) Rare transcripts enriched after SSH procedure: RpCAbr transcript (E) and intracellular globin transcript (F). (G) Abundant transcript in one tissue and rare in other tissue: MVP transcript. (H) Non equally subtracted transcript: cytochrome c oxidase subunit I transcript. The faint bands appearing at a smaller size than expected in some wells are interpreted as non-specific amplification (possible primer dimerization under specific conditions).

Mentions: We only found 3 common cDNAs between the BR-BW and BW-BR libraries and 2 common cDNAs between the TR-BW and BW-TR ones. We were able to assess the degree of successful subtraction for several transcripts by performing regular PCR on subtracted and unsubtracted poly-A cDNA pools. Some results are shown in Fig. 2.


Identification of proteins involved in the functioning of Riftia pachyptila symbiosis by Subtractive Suppression Hybridization.

Sanchez S, Hourdez S, Lallier FH - BMC Genomics (2007)

Typical PCR profiles obtained after amplification of fragments of interesting cDNAs. S = subtracted sample; UN = unsubtracted sample. (A) and (B) Abundant tissue-specific transcripts: exosqueleton β-chitin-binding transcript (A) and galaxin transcript (B). (C) and (D) Transcripts enriched after SSH procedure: chitinase precursor transcript (C) and RpCAtr (D). (E) and (F) Rare transcripts enriched after SSH procedure: RpCAbr transcript (E) and intracellular globin transcript (F). (G) Abundant transcript in one tissue and rare in other tissue: MVP transcript. (H) Non equally subtracted transcript: cytochrome c oxidase subunit I transcript. The faint bands appearing at a smaller size than expected in some wells are interpreted as non-specific amplification (possible primer dimerization under specific conditions).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2175520&req=5

Figure 2: Typical PCR profiles obtained after amplification of fragments of interesting cDNAs. S = subtracted sample; UN = unsubtracted sample. (A) and (B) Abundant tissue-specific transcripts: exosqueleton β-chitin-binding transcript (A) and galaxin transcript (B). (C) and (D) Transcripts enriched after SSH procedure: chitinase precursor transcript (C) and RpCAtr (D). (E) and (F) Rare transcripts enriched after SSH procedure: RpCAbr transcript (E) and intracellular globin transcript (F). (G) Abundant transcript in one tissue and rare in other tissue: MVP transcript. (H) Non equally subtracted transcript: cytochrome c oxidase subunit I transcript. The faint bands appearing at a smaller size than expected in some wells are interpreted as non-specific amplification (possible primer dimerization under specific conditions).
Mentions: We only found 3 common cDNAs between the BR-BW and BW-BR libraries and 2 common cDNAs between the TR-BW and BW-TR ones. We were able to assess the degree of successful subtraction for several transcripts by performing regular PCR on subtracted and unsubtracted poly-A cDNA pools. Some results are shown in Fig. 2.

Bottom Line: Overall, half of the contigs matched records found in the databases with good E-values.Quantitative PCR analyses were congruent with our libraries results thereby confirming the existence of tissue-specific transcripts identified by SSH.Some of the keys to understanding metabolite exchanges may remain in the sequences we could not identify (hypothetical proteins and no similarity found).

View Article: PubMed Central - HTML - PubMed

Affiliation: Equipe Ecophysiologie: Adaptation et Evolution Moléculaires, UMR 7144 CNRS UPMC, Station Biologique, Place Georges Teissier, BP 74, 29682 Roscoff Cedex, France. sanchez@sb-roscoff.fr

ABSTRACT

Background: Since its discovery around deep sea hydrothermal vents of the Galapagos Rift about 30 years ago, the chemoautotrophic symbiosis between the vestimentiferan tubeworm Riftia pachyptila and its symbiotic sulfide-oxidizing gamma-proteobacteria has been extensively studied. However, studies on the tubeworm host were essentially targeted, biochemical approaches. We decided to use a global molecular approach to identify new proteins involved in metabolite exchanges and assimilation by the host. We used a Subtractive Suppression Hybridization approach (SSH) in an unusual way, by comparing pairs of tissues from a single individual. We chose to identify the sequences preferentially expressed in the branchial plume tissue (the only organ in contact with the sea water) and in the trophosome (the organ housing the symbiotic bacteria) using the body wall as a reference tissue because it is supposedly not involved in metabolite exchanges in this species.

Results: We produced four cDNA libraries: i) body wall-subtracted branchial plume library (BR-BW), ii) and its reverse library, branchial plume-subtracted body wall library (BW-BR), iii) body wall-subtracted trophosome library (TR-BW), iv) and its reverse library, trophosome-subtracted body wall library (BW-TR). For each library, we sequenced about 200 clones resulting in 45 different sequences on average in each library (58 and 59 cDNAs for BR-BW and TR-BW libraries respectively). Overall, half of the contigs matched records found in the databases with good E-values. After quantitative PCR analysis, it resulted that 16S, Major Vault Protein, carbonic anhydrase (RpCAbr), cathepsin and chitinase precursor transcripts were highly represented in the branchial plume tissue compared to the trophosome and the body wall tissues, whereas carbonic anhydrase (RpCAtr), myohemerythrin, a putative T-Cell receptor and one non identified transcript were highly specific of the trophosome tissue.

Conclusion: Quantitative PCR analyses were congruent with our libraries results thereby confirming the existence of tissue-specific transcripts identified by SSH. We focused our study on the transcripts we identified as the most interesting ones based on the BLAST results. Some of the keys to understanding metabolite exchanges may remain in the sequences we could not identify (hypothetical proteins and no similarity found). These sequences will have to be better studied by a longer -or complete- sequencing to check their identity, and then by verifying the expression level of the transcripts in different parts of the worm.

Show MeSH
Related in: MedlinePlus