Molecular cloning of the ribosomal P-proteins MgP1, MgP2, MgP0, and superoxide dismutase (SOD) in the mussel Mytilus galloprovincialis and analysis of MgP0 at stress conditions.
The stalk, a characteristic structure of the large ribosomal subunit, is directly involved in the interaction with the soluble factors during translation. In the Mediterranean mussel Mytilus galloprovincialis, the stalk consists of one 32 kDa protein, MgP0, and two smaller, 12 kDa acidic proteins, MgP1 and MgP2, of pI 3.0 and 4.0, respectively, as revealed by analysis of purified ribosomes with electrophoresis and Western blot with a specific monoclonal antibody. Treatment of the ribosomes with alkaline phosphatase showed movement of the bands corresponding to the acidic MgP1 and MgP2 proteins to more basic pH after isoelectrofocusing, implying phosphorylation. The cDNA molecules of M. galloprovincialis ribosomal proteins MgP0, MgP1 and MgP2 and superoxide dismutase (MgSOD) were isolated from a cDNA library or constructed by RT-PCR, cloned in expression vectors and expressed in Escherichia coli. The recombinant proteins were purified with immobilized metal ion affinity chromatography (IMAC) and identified with immunoblotting. Exposure of mussels at cadmium and sorbitol and analysis of gill tissue extracts showed over expression of MgP0 protein.
Kolaiti
Regina-Maria
RM
Department of Genetics and Biotechnology, Faculty of Biology, University of Athens, Panepistimiopolis, 15701 Athens, Greece.
Lucas
Jared M
JM
Kouyanou-Koutsoukou
Sophia
S
D000595:Amino Acid Sequence; D000818:Animals; D002104:Cadmium / Q000494:pharmacology; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D015151:Immunoblotting; D008969:Molecular Sequence Data; D049878:Mytilus / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics*; D012269:Ribosomal Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D012270:Ribosomes / Q000187:drug effects / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013012:Sorbitol / Q000494:pharmacology; D013312:Stress, Physiological / Q000187:drug effects; D013482:Superoxide Dismutase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification
D018076:DNA, Complementary; D012269:Ribosomal Proteins; D002104:Cadmium; D013012:Sorbitol; D013482:Superoxide Dismutase