Novel proteins belonging to the troponin C superfamily are encoded by a set of mRNAs in sea urchin embryos

The properties of several cDNA clones representing a family of mRNAs found in the embryonic ectoderm of Strongylocentrotus purpuratus are described. We have previously shown that these mRNAs (termed Spec for Strongylocentrotus purpuratus ectoderm) accumulate in the presumptive dorsal ectoderm of post-cleavage stage embryos and code for a group of 10 to 12 low molecular weight acidic proteins. We demonstrate here, using antibodies raised against the major Spec proteins, that the proteins are localized in the cytoplasm of dorsal ectoderm cells. Hybridization analysis and DNA sequencing show that the mRNAs coding for these proteins, although all related, can be divided into two subfamilies. Comparison of the translational reading frames of the Spec mRNAs with known protein sequences shows a significant homology with troponin C-related proteins, especially in the calcium-binding domains. We suggest that the Spec proteins are previously uncharacterized members of the troponin C superfamily.     

Regulatory elements from the related spec genes of Strongylocentrotus purpuratus yield different spatial patterns with a lacZ reporter gene.

The Spec1 and Spec2 genes of Strongylocentrotus purpuratus are closely associated with the differentiation of aboral ectoderm. To examine cis-regulatory elements involved in the spatial expression of the Spec genes, we fused the Escherichia coli lacZ gene containing a nuclear targeting signal to 5'flanking DNA plus 5' untranslated leader sequences from Spec1, Spec2a, and Spec2c. All three genes contain 700 bp of highly conserved DNA in their upstream regions, but in Spec1 and Spec2c large insertions interrupt the conserved regions. The Spec-lacZ reporter gene plasmids were microinjected into eggs of S. purpuratus, Lytechinus variegatus, and L. pictus, and beta-galactosidase activity was determined in situ by X-gal staining. The Spec2a-lacZ fusion gene, which contained 1516 bp of 5' flanking DNA and 18 bp of 5' untranslated leader sequence, was preferentially expressed in aboral ectoderm cells in all three species. The Spec1-lacZ fusion gene was expressed in a strikingly different fashion--preferentially in primary and secondary mesenchyme cells, occasionally in aboral ectoderm cells, and less often in oral ectoderm and endoderm cells. The staining pattern was the same in either homologous or heterologous embryos. The Spec2c-lacZ fusion gene, like Spec2a-lacZ, was preferentially expressed in aboral ectoderm, but staining of other cell types was frequently observed. To further delineate sequences required for correct spatial expression, we deleted 800 bp of 5' flanking DNA from the Spec2a-lacZ fusion gene, resulting in a delta Spec2a-lacZ fusion gene that contained only the conserved DNA region. This gene fusion showed preferential expression in aboral ectoderm cells. However, the cell type specificity was not as great as with the parental Spec2a-lacZ plasmid. These experiments implied that the conserved DNA region, associated with all Spec genes examined, was insufficient for complete aboral ectoderm specificity, and suggested that a spatial repressor element existed between -1516 and -697 bp in the 5' flanking DNA of Spec2a.     

Mitochondrial DNA sequence analysis of the cytochrome oxidase subunit II gene from Podospora anserina. A group IA intron with a putative alternative splice site

A 5 kb region of the 95 kb mitochondrial genome of Podospora anserina race s has been mapped and sequenced (1 kb = 10(3) base-pairs). This DNA region is continuous with the sequence for the ND4L and ND5 gene complex in the accompanying paper. We show that this sequence contains the gene for cytochrome oxidase subunit II (COII). This gene is 4 kb in length and is interrupted by a subgroup IB intron (1267 base-pairs (bp) in length) and a subgroup IA intron (1992 bp in length). This group IA intron has a long open reading frame (ORF; 472 amino acid residues) discontinuous with the upstream exon sequence. A putative alternative splice site is present, which brings the ORF into phase with the 5' exon sequence. The 5'- and 3'-flanking regions of the COII gene contain G + C-rich palindromic sequences that resemble similar sequences flanking many Neurospora crassa mitochondrial genes.     

Organization and primary sequence of multiple genes coding for the apopolysialoglycoproteins of rainbow trout

We have shown that the mRNAs for apopolysialoglycoproteins (apoPSGP) of rainbow trout contain various numbers of a repetitive sequence of 39 base-pairs encoding mature apoPSGP, and that this sequence is bordered by highly homologous 5' and 3' regions encoding pre-, pro- and telopeptides. These mRNAs are thought to be transcribed from different genes that constitute a large multiple gene family (more than 100 members). Here, we have determined the structures of several members of the apoPSGP gene family. The results show that two of three genomic DNA fragments contain two independent apoPSGP genes in the same orientation with unrelated sequences intervening. Five characterized genes have essentially the same organization and sequence. Each gene has four exons, and CAAT and TATA sequences were found in the 5'-flanking regions. However, two noteworthy differences were observed among the five genes; a diversity in the number of the 39 base-pair repeats, also observed among the cDNA clones, and a one-base polymorphism in the 39 base-pair repeat, which causes an amino acid change. This polymorphism was not detected among the cDNA clones obtained. The boundary positions of the genes are various and contain no transposon-like structures. The variation in the number of repeats and the absence of a rule for bordering positions of the genes suggest that apoPSGP genes may have been amplified by gene duplications, unequal recombination, and selection of chromosomes having larger numbers of apoPSGP genes.     

Unusual sequence conservation in the 5′ and 3′ untranslated regions of the sea urchin spec mRNAs

Summary The Spec1 and Spec2 mRNAs (Strongylocentrotus purpuratus ectoderm mRNAs) represent a small gene family that encodes 10–12 members of the troponin C superfamily of calcium-binding proteins. These mRNAs and proteins accumulate in the aboral (dorsal) ectoderm of sea urchin embryos and larvae. Using genomic and cDNA clones, we have compared the sequences of four Spec mRNAs: Spec1, Spec2a, Spec2c, and Spec2d. The mRNAs all have at least 120 bases of 5 untranslated leader, approximately 450 bases of open reading frame, and 900 bases (Spec1) or 1250 bases (Spec2a, 2c, 2d) of 3 untranslated trailer. Unexpectedly, when long stretches of 5 untranslated regions or 3 untranslated regions are compared to one another, they are found to be less divergent than the protein-coding regions. Comparing Spec2d, the most divergent member of the family, with the other Spec mRNAs shows that while the protein-coding regions are 60–62% matched, the untranslated regions are greater than 80% matched. Comparisons among Spec1, Spec2a, and Spec2c demonstrate similar but less dramatic conservation of untranslated regions. Our data imply that the Spec gene family has evolved differently from most gene families, with mutations accumulating most rapidly in intron regions, less rapidly in protein-conding regions, and least rapidly in 5 and 3 untranslated regions.     

Tandem duplication and divergence of a sea urchin protein belonging to the troponin C superfamily

The Spec1 and Spec2 proteins of the sea urchin Strongylocentrotus purpuratus are related to calmodulin, troponin C, and myosin light chains by sequence similarity in their four calcium binding domains. These domains, the EF-hands, are distinct helix-loop-helix structures of about 40 amino acids. The Spec1 and Spec2 genes are expressed specifically in aboral ectoderm cells of the developing embryo; however, the function of the Spec proteins in these cells is unknown. To find conserved regions of the proteins that might be important for structure and function, Spec homologues from Lytechinus pictus, a distantly related sea urchin, were sought. L. pictus embryos do not synthesize detectable amounts of the 14,000-17,000-Da Spec proteins as determined by two-dimensional gel electro-phoresis, but do synthesize three 34,000-Da proteins that cross-react with Spec1 antibodies and display a similar ontogenetic pattern of expression. cDNA clones were isolated by hybridization to a synthetic oligonucleotide corresponding to the EF-hand. One clone, LpS1, encodes an mRNA with developmental properties like those of the S. purpuratus Spec mRNAs. However, LpS1 contains an open reading frame for a protein of 34,000 Da rather than 17,000 Da, and antibodies raised against part of the LpS1 reading frame demonstrate that LpS1 encodes a 34,000-Da protein in L. pictus embryos. The sequence of LpS1 reveals the presence of eight EF-hand domains, which share structural homology with the Spec1 or Spec2 EF-hands; however, little else in the protein sequence is conserved. The results support the hypothesis that the LpS1 gene arose from a duplication of an ancestral Spec gene and that the overall structural features of the Spec family of proteins are more conserved than the amino acid sequences.