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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 Mol├ęculaires, 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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Relative expression levels of ribosomal RNA 16S, ccox I, ATPF1, Cathepsin, RpCAbr, RpCAtr, and MVP transcripts in the branchial plume, trophosome and body wall tissues. For each transcript, the calibrator tissue was chosen as the tissue with the higher expression: the branchial plume was the calibrator for ribosomal RNA 16S, ccox I, Cathepsin, RpCAbr and MVP amplifications, the trophosome was the calibrator for RpCAtr and ATPF1 amplifications. The number of tissue replicates (n) ranges from 3 to 4, and corresponds to the number of intra-individual tissue pairs we had.
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Figure 3: Relative expression levels of ribosomal RNA 16S, ccox I, ATPF1, Cathepsin, RpCAbr, RpCAtr, and MVP transcripts in the branchial plume, trophosome and body wall tissues. For each transcript, the calibrator tissue was chosen as the tissue with the higher expression: the branchial plume was the calibrator for ribosomal RNA 16S, ccox I, Cathepsin, RpCAbr and MVP amplifications, the trophosome was the calibrator for RpCAtr and ATPF1 amplifications. The number of tissue replicates (n) ranges from 3 to 4, and corresponds to the number of intra-individual tissue pairs we had.

Mentions: Relative expression levels were calculated between the different tissues of a whole organism after normalization of the transcripts amplifications with the 18S reference gene. The results from the analysis of several individuals are shown in Fig. 3. The 16S ribosomal gene has 7.5-fold and 10-fold higher expression levels in the branchial plume compared to the trophosome and the body wall, respectively. The ccox I and ATP synthase F1 transcripts were equally present in the branchial plume and trophosome tissues but comparatively less abundant in the body wall tissue (about 16-fold and 43-fold, respectively). The new CA sequence (RpCAbr) is preferentially expressed in the branchial plume tissue compared to the trophosome (1,000-fold less expression) and the body wall (109-fold less expression) tissues. On the opposite, the RpCAtr transcript was more abundant in the trophosome tissue than in the branchial plume (12-fold less expression) and the body wall (2,500-fold less expression) tissues. Relative quantification analyses also showed that cathepsine L-like genes are also up-regulated in the branchial plume tissue compared to the trophosome (about 4-fold less expressed) and the body wall (about 7-fold less expressed) tissues of the worm. The MVP transcript was nearly 10-fold more abundant in the branchial plume than in the body wall tissue but we could not detect it in the trophosome tissue total cDNA.


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

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

Relative expression levels of ribosomal RNA 16S, ccox I, ATPF1, Cathepsin, RpCAbr, RpCAtr, and MVP transcripts in the branchial plume, trophosome and body wall tissues. For each transcript, the calibrator tissue was chosen as the tissue with the higher expression: the branchial plume was the calibrator for ribosomal RNA 16S, ccox I, Cathepsin, RpCAbr and MVP amplifications, the trophosome was the calibrator for RpCAtr and ATPF1 amplifications. The number of tissue replicates (n) ranges from 3 to 4, and corresponds to the number of intra-individual tissue pairs we had.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Relative expression levels of ribosomal RNA 16S, ccox I, ATPF1, Cathepsin, RpCAbr, RpCAtr, and MVP transcripts in the branchial plume, trophosome and body wall tissues. For each transcript, the calibrator tissue was chosen as the tissue with the higher expression: the branchial plume was the calibrator for ribosomal RNA 16S, ccox I, Cathepsin, RpCAbr and MVP amplifications, the trophosome was the calibrator for RpCAtr and ATPF1 amplifications. The number of tissue replicates (n) ranges from 3 to 4, and corresponds to the number of intra-individual tissue pairs we had.
Mentions: Relative expression levels were calculated between the different tissues of a whole organism after normalization of the transcripts amplifications with the 18S reference gene. The results from the analysis of several individuals are shown in Fig. 3. The 16S ribosomal gene has 7.5-fold and 10-fold higher expression levels in the branchial plume compared to the trophosome and the body wall, respectively. The ccox I and ATP synthase F1 transcripts were equally present in the branchial plume and trophosome tissues but comparatively less abundant in the body wall tissue (about 16-fold and 43-fold, respectively). The new CA sequence (RpCAbr) is preferentially expressed in the branchial plume tissue compared to the trophosome (1,000-fold less expression) and the body wall (109-fold less expression) tissues. On the opposite, the RpCAtr transcript was more abundant in the trophosome tissue than in the branchial plume (12-fold less expression) and the body wall (2,500-fold less expression) tissues. Relative quantification analyses also showed that cathepsine L-like genes are also up-regulated in the branchial plume tissue compared to the trophosome (about 4-fold less expressed) and the body wall (about 7-fold less expressed) tissues of the worm. The MVP transcript was nearly 10-fold more abundant in the branchial plume than in the body wall tissue but we could not detect it in the trophosome tissue total cDNA.

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 Mol├ęculaires, 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