A cDNA from the sea urchin Strongylocentrotus purpuratus encodes a 624 amino acid polypeptide (WEE1S.purp) with a high degree of similarity to the Mik1 and Wee1 protein tyrosine kinases. These kinases act as negative regulators of mitosis by inactivating cyclin-dependent kinases (CDK). Wee1 activity varies during the cell-cycle, and is generated only when required. The pattern of WEE1S.purp mRNA expression was examined temporally and spatially in sea urchin embryos. Only a trace amount of WEE1S.purp mRNA is present in the egg and through the fifth cell cycle post-fertilization. During the next three cycles to the mid-blastula stage, its concentration rises transiently to 2.5 x 10(4) transcripts per embryo. Its developmental profile during this early period is the inverse of that reported for cyclin mRNAs, which are at a high level in the egg and through the fifth cell cycle, then decline upon further development. WEE1S.purp mRNA in the gastrula and pluteus stages becomes restricted to cells engaged in DNA replication, including the endoderm (gut), oral ectoderm, and arm rudiments. It is absent from the aboral ectoderm, which lacks cycling cells. In the pluteus larva of the species Lytechinus pictus, WEE1 mRNA was detected in the arm rudiments during cellular proliferation and arm elongation, but not after the completion of the arms. Putative regulatory motifs in the sea urchin Wee1-like cDNA suggest a capacity for rapid turnover of both its mRNA and protein: The WEE1S.purp mRNA 3' UTR contains 13 AUUUA pentamers, which have been characterized as determinants of mRNA lability; and the N-terminal domain of the predicted WEE1S.purp polypeptide is enriched in S/TP-containing, potential kinase-target sites, as well as high-value "PEST' sequences, associated with protein lability. The developmental appearance of WEE1S.purp mRNA may coincide with the introduction of a gap phase in the cell cycle. Its spatial pattern during embryogenesis appears to reflect distinct programs of regulated cell cycling in differentiating tissues.

The SpMTA metallothionein gene of the sea urchin Strongylocentrotus purpuratus is regulated developmentally, histospecifically, and by heavy-metal induction. The mentally scheduled (35, 50) and histospecifically regulated (34). Since its expression is restricted to the aboral ectoderm in sea urchin gastrulae and pluteus larvae, cultured in either the absence or presence of added heavy metal ions, metal induction does not override the histospecificity but appears, indeed, to be linked to it (34). Positive regulatory elements have been implicated in several genes expressed in the sea urchin embryo, and these together with negatively operative spatial-control elements have been identified in genes, such as those for cytoskeletal CyIIIa actin (48) and Spec 1 and Spec 2 troponins (16), which display the same aboral ectoderm specificity as SpMTA (34). Only one such element is indicated in the previously sequenced 0.6 kb of the 5' flank of the SpMTA gene (20). However, we have identified a cluster of elements in the first intron of this gene with homology to the above-mentioned positive and spatial-control elements. Moreover, the absence of this intronic region from another MT gene, SpMTB1, which is not aboral ectoderm specific (34), suggests that it should be studied as a potentially specific regulator of SpMTA gene expression.To appreciate the full extent of promoter involvement in metal-induced expression, as well as the regulative participation of the first intron, we have obtained an additional 5'-flanking sequence and examined the activities in transgenic embryos of reporter gene constructs containing portions of 5'-flanking and intronic regions. Our observations indicate that the SpMTA gene is functionally bipartite, insofar as the 5' flank promotes metal-regulated transcription, while the first intron amplifies this activity. MATERIALS AND METHODSSequencing and preparation of plasmids. The SpMTA MT X206 clone from an S. purpuratus genomic DNA library (20) was the source of a previously restriction site-mapped subclone to be sequenced. A 1-kb region between AccI and PstI on May 9, 2018 by guest

The SpMTA metallothionein (MT) gene of the sea urchin Strongylocentrotus purpuratus is restricted in its expression to the aboral ectoderm in gastrulae and pluteus larvae. The proximal 1.6 kb of the 5'-flanking region together with the 1.12-kb first intron of the SpMTA gene are sufficient for its correct cell-type specific expression in transgenic embryos. This restricted spatial expression is largely eliminated by deletion of an interior 405-bp region in the intron. Within this region is a 295-bp, genomically repetitive, transposon-like segment (Nemer et al., 1993), containing several sequence motifs highly homologous to posited regulatory elements in the promoters of other genes (Thiebaud et al., 1990). The P3A and P5 sites in this apparent regulatory cassette were shown through competition to bind with relatively high affinities the same nuclear factors, bound by their counterpart sites in the CyIIIa actin promoter.

A region in the first intron of a metallothionein-encoding gene of the sea urchin Strongylocentrotus purpuratus (SpMTA gene) regulates its 5' promoter activity. Within this region is a 290-bp cassette of six sequence motifs that are present in other genes in this species and posited to operate as regulatory elements. The cassette, present at high multiplicity in the genome, was used to screen genomic DNA clones. Of these, six diverse individuals were partially sequenced and found to have segments 94% identical to the 290-bp cassette in the SpMTA gene. Their next 80 bp diverged from the SpMTA sequence but were highly identical among the six non-SpMTA clones and contained an additional regulatory motif. These diverse clones thus contained 370-bp cassettes of an overall 94% sequence identity and an apparent content of seven regulatory elements. The regulatory cassettes were transposon-like, insofar as the termini of the highly identical regions consisted of 24-to 25-bp inverted repeats, bracketed by 6-to 9-bp direct repeats in the divergent regions. In addition to being in transcripts of the SpMTA intron, the cassette was found in other sea urchin embryo poly(A)+ RNAs, in eggs and embryos, and enriched in pluteus ectoderm. The cassette sequence was present in moderate abundance in transcripts in both sense and antisense orientation. We report the presence of a transposon-like cassette of regulatory elements that is also represented in RNA, which potentially could function differently from previously described transposons.A DNA segment appearing as an insert in the interior of the first intron of the MTA metallothionein (MT) gene (1) of the sea urchin Strongylocentrotus purpuratus (SpMTA gene) distinguishes it from the first intron of another, substantially homologous MT gene in this species, SpMTB1 (2). The interior of the SpMTA intron is largely responsible for the high degree of activity of this gene (3), in contrast to the low-level activity (4) of the SpMTB, gene. This specifically SpMTA DNA segment may be functionally similar to inserted DNA elements, which in various situations can elicit changes in gene regulation (5,6), become, themselves, transcriptionally regulated (7), or produce disruptive and deleterious effects (8,9). Through such an intronic insert, the SpMTA gene may have been recruited evolutionarily into a specifically regulated set, to which the other MT genes in this species do not belong. This insert is particularly interesting because it contains within a stretch of0.3 kb several sequence motifs highly identical to sites in several other genes (10) and specifically implicated as regulatory elements in CyIla (11,12), an actin-encoding gene that has the same aboral ectodermal specificity of expression (13) as the SpMTA gene (2).The premise that this 0.3-kb intronic region, enriched in putative regulatory sites, was indeed inserted into an evolutionary progenitor of the SpMTA gene has led us here to The publication costs of this article were defrayed in part by page charge payment. This a...

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