The stringlike genes of the limpet Patella vulgata.

As a first step in analyzing the function of a cdc25 homolog during the embryonic development of Patella vulgata (Pv), genomic clones encoding these stringlike proteins (Stl) were isolated and characterized. These clones belong to four groups which are derived from different regions of the Pv genome. As the sequences of Stl genes from two of these groups are almost identical, we suggest that these genes represent copies of the same gene. The Stl3 gene, which has been analyzed in detail, consists of four exons separated by three introns. Its sequence encodes a 250-amino-acid protein with a calculated weight of 28 kDa. The Stl protein contains regions conserved in all other cdc25 proteins. Stl messengers are not stored maternally in Pv oocytes and Stl transcription only starts after the first embryonic cleavages.

Localization and quantification of cyclin A and B mRNA during the embryonic development of Patella vulgata.

As the first five cleavages of the Patella vulgata embryo are synchronous, they are well suited to determine the mRNA level of cyclin A and B genes in an embryo. During the third and fourth cleavage cycle the quantity of A and B mRNA is regulated in a cell-cycle-dependent way, reaching a high level between cleavages and a lower level just after mitosis. This implies that transcription of the cyclin genes occurs before the overall transcription increases directly after the fifth cleavage. During the first cleavages cyclin A and B mRNA is localized in distinct parts of the cytoplasm. Between two successive cell devisions it is found as a crescent-shaped domain at the peripheral side of the nucleus. At cytokinesis it is present between two separating nuclei and at newly formed cell membranes. At the fifth cleavage this localization disappears. Changes in the expression pattern of cyclin A and B may be expected after the fifth cleavage, when the first cells become arrested in cell division and differentiate. The mechanism causing cell division arrest of these primary trochoblasts is still unknown. Cell division arrest caused by the absence of cyclin A and/or B mRNA could be conditional for further differentiation. However, a decrease in cyclin A and B mRNA level in the trochoblasts is not detectable until 4 h after their last division. Later in development no cyclin A and B mRNA can be detected in these cells, whereas cyclin A and B mRNA is present in other cells of the embryo. Thus, the absence of cyclin A and B mRNA in primary trochoblasts, and in the later differentiating secondary and accessory trochoblasts is not obligatory for cell division arrest or cell differentiation.

The role of cyclins in the maturation of Patella vulgata oocytes.

We have cloned and sequenced the cDNAs encoding Patella vulgata cyclins A and B. The cDNA clones contain an open reading frame of 426 and 408 amino acids respectively, which present similarity with cyclins from other species. Cyclin A and B RNAs are present as polyadenylated and non-polyadenylated RNA in prophase oocytes and are completely polyadenylated in metaphase I. During the first cleavages after fertilization the level of cyclin A and B mRNAs is high and drops when the free swimming stage is reached. Using p13suc1-Sepharose bead precipitation we demonstrate that cyclin synthesis is triggered during maturation and that inhibition of protein synthesis makes the cyclins disappear rapidly from the metaphase I oocytes, which shift to interphase condition. By microinjecting antisense oligonucleotides into metaphase I oocytes, we demonstrate that in vivo ablation of cyclin A and B messengers together gives the same result, whereas microinjection of only one oligonucleotide does not show any effect.