Richard M. Showman

Antibodies to a fusion protein identify a cDNA clone encoding msp130, a primary mesenchyme-specific cell surface protein of the sea urchin embryo

In this report we identify a 130-kDa protein encoded by a sea urchin primary mesenchyme-specific cDNA clone, 18C6. The cDNA clone has been partially sequenced, and an open reading frame has been identified. A portion of this open reading frame has been expressed as a beta-galactosidase fusion protein in Escherichia coli, and antibodies to the fusion protein have been generated. These antibodies recognize a 130-kDa protein localized at the surface of primary mesenchyme cells and designated msp130. This is demonstrated to be the same 130-kDa protein recognized by the primary mesenchyme-specific monoclonal antibody B2C2, which recognizes a post-translational modification of the protein. RNA gel blots show that the transcript encoding msp130 is undetectable in egg RNA or 16-cell RNA but can be first detected in premesenchyme blastula embryos. The transcript accumulates significantly after primary mesenchyme cell ingression. Analysis of the expression of msp130 by indirect immunofluorescence staining of embryos and by immunoblots using fusion protein antibodies shows that the msp130 protein is first detectable soon after primary mesenchyme cell ingression.

Genes for Chromosomal Proteins Expressed before and after Meiosis

Cloned gene sequences have been isolated for two testis-specific chromosomal proteins, one of which, histone (H1t), appears during meiosis, whereas the other, transition protein 1 (TP1), appears only during the later steps of spermatid development. Aspects of the regulation of each gene have been examined. In the case of H1t, analysis of its promoter region shows that it contains excellent matches to each of the four sequence homologies identified for the usual somatic H1 variants, so that the factor(s) that restrict H1t expression to spermatocytes remain a mystery. In the case of TP1, a cDNA clone allowed identification of its message by Northern blots as well as by in situ hybridization. The message appears postmeiotically in late round spermatids but is translationally repressed until the spermatid nucleus begins to condense.

Temporal and spatial expression of embryonic alkaline phosphatase activity in the surf clam, Spisula solidissima

The temporal appearance and spatial distribution of alkaline phosphatase (EC 3.1.3.1) have been examined during the development of the surf clam, Spisula solidissima. Enzyme activity was detectable at low levels from fertilization onward with a sharp linear increase in activity occurring at the trochophore stage, 14 h post-fertilization. Histochemical localization of enzyme activity at the light microscope level showed the increased activity associates initially with a limited number of large macromeres and is progressively restricted to the cells forming the tip of the growing archenteron. Electron microscopy shows the activity is restricted to the luminal and lateral plasma membrane of these cells.

Maternal stores of α subtype histone mRNAs are not required for normal early development of sea urchin embryos

SummaryThe ability to specifically delete the store of maternal α-subtype histone mRNAs stored in the egg pronucleus has allowed us to examine the role of this major fraction of the maternal mRNA in the early development of the sea urchinStrongylocentrotus purpuratus. The egg nucleus was removed by centrifugation, and the resulting enucleate half eggs were fertilized. These haploid andromerogones lacked any stored α-subtype histone mRNAs. However, when grown in parallel with control embryos, they showed identical cleavage cycles, cell numbers, and patterns of cell differentiation. Measurements of the amount of α-histone mRNA in these andromerogones showed that there was no premature synthesis of α-histone mRNAs to compensate for the deleted maternal pool. Instead embryonic synthesis was normal in timing of initiation and duration. the ability of these embryos to develop into highly differentiated larvae without their maternal α-subtype histone mRNA pool suggests that this pool is not a critical component of early development per se. This suggestion is strengthened by the observation that the primitive sea urchinEucidaris tribuloides naturally lacks this maternal histone mRNA store. Evolutionary implications are discussed.

Na+, K+-ATPase, Mg2+-ATPase and Ca2+-ATPase activity during arm regeneration in the starfish Asterias forbesi

Abstract 1. 1. The starfish, Asterias forbesi lacks a ouabain-sensitive, Mg 2+ -dependent Na + , K + -ATPase but possesses Mg 2+ -ATPase. 2. 2. This Mg 2+ -ATPase activity does not change during arm regeneration. 3. 3. The starfish also lacks a high-affinity Ca 2+ -ATPase, but possesses a low-affinity Ca 2+ -ATPase. 4. 4. The low-affinity Ca 2+ -ATPase activity increases 7 days after autotomy. Levels of activity return to those of control non-regenerating individuals by 20 days post-autotomy, the time at which newly formed ossicles are first visible.

Translation of maternal histone mRNAs in sea urchin embryos: A test of control by 5′ cap methylation☆

Recent results have demonstrated the occurrence of mRNA cap methylation in the sea urchin embryo following fertilization. It has been suggested that this methylation event is responsible for the translational activation of maternal histone mRNAs in these embryos. We have used aphidicolin, an effective inhibitor of both DNA synthesis and cap methylation in cleavage stage sea urchin embryos, to examine the relationship between cap methylation and translation. At 5 micrograms/ml, a dose which rapidly abolishes DNA replication and blocks cleavage, we note no effect on recruitment or translation of maternal alpha-subtype histone mRNAs. This suggests that a postfertilization cap methylation event is not critical to the process of regulation of the translation of stored alpha-subtype histone mRNAs.

Uptake of free amino acids by the ophiuroid Microphiopholis gracillima (Stimpson) (Echinodermata) during disc regeneration

Abstract 1. 1. Intact and regenerating brittlestars (Microphiopholis gracillima) take up several amino acids from artificial seawater, and show significant increases in uptake with food deprivation. 2. 2. There were no differences in total amino acid uptake rates (determined by high performance liquid chromatography) between intact and regenerating animals. 3. 3. No increases in the gross rate of leucine uptake were apparent in either group over time; however, glycine uptake increased significantly. 4. 4. Catabolism of leucine and glycine, as evidenced by labeled CO2 release, was highest among intact animals that had been held for several days. 5. 5. Dissolved leucine appears to be rapidly catabolized, while glycine tends to remain in tissues.