Characterization and expression of a gene encoding a 30.6-kDa Strongylocentrotus purpuratus spicule matrix protein.
We describe here the isolation and characterization of several cDNA clones that encode a single 30.6-kDa Strongylocentrotus purpuratus spicule matrix protein designated SM30. The clones were isolated by screening a lambda gt11 cDNA library with a rabbit polyclonal antiserum raised against S. purpuratus total spicule matrix proteins. DNA sequencing reveals that the SM30 protein is acidic. RNA blot analysis shows that the cDNAs hybridize to a single 1.8-kb transcript and that there is a sharp increase in the SM30 transcript levels at middle to late mesenchyme blastula stage. SM30 transcript levels remain high through the 3-day pluteus stage. In situ hybridization analysis indicates that, within the embryo, SM30 transcript accumulation is restricted to the primary mesenchyme cells. Quantitations of SM30 transcript levels show that by the prism stage there are about 29,000 SM30 transcripts present per embryo, which averages to approximately 480 transcripts per primary mesenchyme cell. Additionally, RNA blot analysis of total RNA isolated from adult tissues shows that SM30 mRNA accumulates exclusively in mineralized tissues. These findings taken together strongly suggest that the gene corresponding to the SM30 cDNAs does in fact encode a spicule matrix protein.
George
N C
NC
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Killian
C E
CE
Wilt
F H
FH
D000375:Aging / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D003598:Cytoskeletal Proteins / Q000235:genetics*; D004625:Embryo, Nonmammalian / Q000378:metabolism; D005786:Gene Expression Regulation; D015723:Gene Library; D016018:Least-Squares Analysis; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D012313:RNA / Q000032:analysis / Q000302:isolation & purification; D015347:RNA Probes; D015183:Restriction Mapping; D012617:Sea Urchins / Q000235:genetics*
D003598:Cytoskeletal Proteins; D015347:RNA Probes; C071119:SM30 protein, Strongylocentrotus purpuratus; D012313:RNA