Materials and methods

RT-PCR

RT-PCR was carried out described in the manuscript. The primers used for Hydra viridis actin were same as mentioned in ‘Expression analysis by RT-PCR’. Chlorella actin was amplified with: 5˘-TGCGATCCAGGCGGTGCTGTCTC-3˘ and 5˘-GAACATGGTGGTGCCGCCCGACA-3˘.

Southern analysis

Preparation of genomic DNA from Hydra viridis

Genomic DNA was isolated from washed animals. Polyps were pelleted and rinsed with 0.53 PBS. Cells were supended by tryptic digestion and pelleted. After lysis (0.2·mol·l^–1 Tris-HCl, pH 8.5, 0.1·mol·l^–1 EDTA, 0.2% SDS) DNA was treated with proteinase K (100·µg·ml^–1) for 90·min at 60°C. After inaktivation of proteins with DEPC DNA was precipitated solubilized in TE-Buffer and RNase treated prior to HindIII digestion.

Preparation of genomic DNA from Chlorella

DNA from isolated symbiotic algae and free-living Chlorella 211-11b was purified as described previously (Kloppstech and Schweiger, 1976). DNA was RNase digested afterwards in order to remove RNA traces. Algal DNA was Csp6I digested.

Electrophoresis and blotting

Samples were loaded on a 1 % Agarose Gel and ran for 6 hours, depurinated in 0.2·mol·l^–1 HCL for 15 min, denatured (1.5M NaCl; 0.5M NaOH) and neutralized (1·mol·l^–1 Tris, pH 7.4; 1.5·mol·l^–1 NacCl). After equlibration in 203 SSC, DNA was transferred on a Nylon Membrane, UV-cross linked and baked at 80°C for 45 min.

Hybridization

Hybridization was carried out as described for northern analysis. The probe was amplified from Hydra viridis cDNA with the following set of primers: 5˘-GTACAATGGTACCAGTAATTTG-3˘ and 5˘-CTGTAGTAGGCGTTATCAAG-3˘.

References

Kloppstech K. and Schweiger, H. G. (1976). In vitro translation of poly (A) RNA from Acetabularia. Cytobiologie 13, 394-400.

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig.·S1. RT-PCR demonstrating complete separation of symbionts from Hydra prior to isolation of RNA. RT-PCR was carried out on cDNA obtained from symbiotic (lanes a–c) and aposymbiotic (lanes d–f) Hydra viridis polyps as well as on cDNA from symbiotic Chlorella (lanes g–i). Lanes (a) and (d), RT-PCR using primers specific for Hydra viridis actin; lanes (b), (e) and (h), water control; lanes (c) and (f), RT-PCR using primers specific for Chlorella actin. Lane (g), RT-PCR on Chlorella cDNA using primers specific for Hydra viridis actin; lane (i) RT-PCR on Chlorella cDNA using primers specific for Chlorella actin. Number of cycles used is indicated on each lane. Since no traces of Chlorella actin could be detected in cDNA from Hydra and since Hydra actin could not be amplified from Chlorella cDNA, symbionts were obviously separated efficiently from Hydra tissue prior to RNA preparation.

• Supplemental Figure 2 -

  Fig.·S2. Sequences of HvAPX1 cDNA and predicted protein. The complete sequence of the HvAPX1 cDNA and the amino acid sequence of the single large open reading frame are presented. Grey letters indicate untranslated regions. A putative spliced leader sequence is underlined. Black letters indicate the coding region. The plant peroxidase domain is highlighted by bold black letters. A signal peptide is indicated by blue letters.

• Supplemental Figure 3 -

  Fig.·S3. The HvAPX1 gene is present in the Hydra viridis genome and cannot be detected in DNA prepared from Chlorella. Southern blot of genomic DNA preparations of symbiotic Hydra viridis (s), aposymbiotic Hydra viridis (a), symbiotic Chlorella (s) and free-living Chlorella 211-11b (f). Hydra DNA was digested with HindIII and Chlorella DNA with Csp6I. Hybridization was carried out using a radioactively labelled HvAPX1 probe of 685·bp.