Neuron-specific expression of a synaptotagmin gene in the sea urchin Strongylocentrotus purpuratus.
Interest in chordate evolution has emphasized a need for a better understanding of the comparative neuroanatomy of invertebrate deuterostomes. However, molecular and genetic approaches to neurobiological studies in these groups are hampered by a lack of neuron-specific molecular markers. A monoclonal antibody, 1E11, is neuron specific and is useful in identification of neural structures in larvae and adults of echinoderms, hemichordates, and urochordates. To identify a neuron-specific gene product, we have characterized the antigen recognized by 1E11. In immunoblots and immunoprecipitations of neural tissue from adult Strongylocentrotus purpuratus, 1E11 recognizes a 57-kDa band. Tandem mass spectrometry of trypsin digests of the 57-kDa band permitted peptide mass mapping and sequencing of five peptides. All of the sequenced peptides, and 12 additional mass-mapped peptides, are found within the open reading frame of a cDNA encoding synaptotagmin B (Sp-SynB). In situ RNA hybridizations with synaptotagmin B probes with S. purpuratus larvae reveal a pattern of expression that is similar to that revealed by the antibody 1E11. Antibodies produced against a bacterially expressed Sp-SynB protein recognize a 57-kDa protein and colocalize with 1E11. When a full-length Sp-SynB cDNA is expressed in chicken embryonic cells, the cells become immunoreactive to 1E11. We conclude that synaptotagmin B is a gene expressed in neurons that has conserved epitopes in other invertebrate deuterostomes.
Burke
Robert D
RD
Department of Biology and Biochemistry, University of Victoria, Victoria, British Columbia V8W 3N5, Canada. rburke@uvic.ca
Osborne
Lisa
L
Wang
Diana
D
Murabe
Naoyuki
N
Yaguchi
Shunsuke
S
Nakajima
Yoko
Y
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000378:metabolism; D015153:Blotting, Western / Q000379:methods; D002642:Chick Embryo; D004330:Drosophila; D015870:Gene Expression / Q000502:physiology*; D018507:Gene Expression Regulation, Developmental / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007150:Immunohistochemistry / Q000379:methods; D047468:Immunoprecipitation / Q000379:methods; D017403:In Situ Hybridization / Q000379:methods; D007814:Larva; D013058:Mass Spectrometry / Q000379:methods; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D009474:Neurons / Q000502:physiology*; D010449:Peptide Mapping / Q000379:methods; D010802:Phylogeny; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012617:Sea Urchins; D050857:Synaptotagmins / Q000235:genetics / Q000378:metabolism*
D000906:Antibodies; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D050857:Synaptotagmins; D049452:Green Fluorescent Proteins