Molecular cloning of five individual stage- and tissue-specific mRNA sequences from sea urchin pluteus embryos.

Abstract

Five developmentally regulated sea urchin mRNA sequences which increase in abundance between the blastula and pluteus stages of development were isolated by molecular cloning of cDNA. The regulated sequences all appeared in moderately abundant mRNA molecules of pluteus cells and represented 4% of the clones tested. There were no regulated sequences detected in the 40% of the clones which hybridized to the most abundant mRNA, and the screening procedures were inadequate to detect possible regulation in the 20 to 30% of the clones presumably derived from rare-class mRNA. The reaction of 32P[cDNA] from blastula and pluteus mRNA to dots of the cloned DNAs on nitrocellulose filters indicated that the mRNAs complementary to the different cloned pluteus-specific sequences were between 3-and 47-fold more prevalent at the pluteus stage than at the blastula stage. Polyadenylated RNA from different developmental stages was transferred from electrophoretic gels to nitrocellulose filters and reacted to the different cloned sequences. The regulated mRNAs were undetectable in the RNA of 3-h embryos, became evident at the hatching blastula stage, and reached a maximum in abundance by the gastrula or pluteus stage. Certain of the clones reacted to two sizes of mRNA which did not vary coordinately with development. Transfers of RNA isolated from each of the three cell layers of pluteus embryos that were reacted to the cloned sequences revealed that two of the sequences were found in the mRNA of all three layers, two were ectoderm specific, and one was endoderm specific. Four of the regulated sequences were complementary to one or two major bands and one to at least 50 bands on Southern transfers of restriction endonuclease-digested total sea urchin DNA.The sea urchin embryo appears to be one of the most accessible developmental systems for studying the molecular events of cell type determination during early embryogenesis. After fertilization, eggs divide into a number of cells whose developmental fate is fixed somewhat before they differentiate into the various cell types. Most of the cells in the sea urchin embryo contribute to the three layers of the relatively simple pluteus larva: the ectodermal covering, the skeletal-forming mesenchyme, and the gut. These differentiated tissues function during the 1 to several months of larval life. The actual sea urchin emerges at metamorphosis after it has grown from a few cells set aside during embryogenesis for its development.Despite the simplicity of the system and the numerous molecular studies on sea urchin embryos, these larval tissues have not received much attention as specialized cells synthesizing cell type-specific proteins. The major reason has been that there is no evidence of very abundant With the advent of molecular cloning, it has become possible to define, isolate, and study genes coding for proteins produced only in limited abundance, including putative specialized proteins of the three cell layers of the pluteus. Clones of mRNA and genomic sequences which code for sev...

The cellular RNA-binding protein EAP recognizes a conserved stem-loop in the Epstein-Barr virus small RNA EBER 1.

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Phylogeny and expression of axonemal and cytoplasmic dynein genes in sea urchins.

“…This region is highly conserved among animal tubulins (Cleveland and Sullivan, 1985). The actin probe was a 1.7-kilobase (kb) BamHl restriction fragment of the sea urchin Tg616 actin gene kindly supplied by Dr. Tom Humphreys' laboratory, University of Hawaii (Fregien et al, 1983). Radiolabelled probes were prepared with [a-32P]dCTP using random hexamer primers and appropriate cDNA as template.…”

Progressively restricted expression of a homeo box gene within the aboral ectoderm of developing sea urchin embryos.

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“…DNAs were digested with restriction endonucleases (15 wg/lane), electrophoresed on 0.8% horizontal agarose gels (Fregien et al 1983), and transferred to nitrocellulose filters (Southern 1975). After baking, filters were hybridized with nick-translated template 1, washed as described previously , covered with plastic wrap, and exposed to Kodak XAR-5 film with intensifying screens at -70~ for 18 hr.…”