Cloning and expression of new receptors belonging to the immunoglobulin superfamily from the marine sponge Geodia cydonium

 A cDNA encoding a receptor tyrosine kinase (RTK) was previously cloned and expressed from the marine sponge (Porifera) Geodia cydonium. In addition to the two intracellular regions characteristic for RTKs, two immunoglobulin (Ig)-like domains are found in the extracellular part of the sponge RTK. In the present study it is shown that no further Ig-like domain is present in the upstream region of the cDNA as well as of the gene hitherto known from the sponge RTK. Two different full-length cDNAs have been isolated and characterized in the present study, which possess two Ig-like domains, one transmembrane segment, and only a short intracellular part, without a TK domain. The two deduced polypeptides were preliminarily termed sponge adhesion molecules (SAM). The longer form of the SAM, GCSAML, encodes a deduced aa sequence, GCSAML, which comprises in the open reading frame 505 amino acids (aa) and has a calculated M _r of 53911. The short form, GCSAMS, has 313 aa residues and an M _r of 33987. The two Ig-like domains in GCSAML and GCSAMS are highly similar to the corresponding Ig-like domains in the RTKs from G. cydonium; the substitutions on both the aa and nt level are restricted to a few sites. Phylogenetic analyses revealed that the Ig-like domain 1 is similar to the human Ig lambda chain variable region, while the Ig-like domain 2 is related more closely to the human Ig heavy chain variable region. Transplantation experiments (autografting) were performed to demonstrate that the level of expression of the two new genes, GCSAML and GCSAMS, is upregulated during the self/self fusion process. Immunohistochemical analyses using antibodies raised against the two Ig-like domains demonstrate a strong expression in the fusion zone between graft and host. This finding has been supported by northern blotting experiments that revealed that especially GCSAML is strongly upregulated after autografting (up to 12-fold); the expression of GCSAMS reaches a value of 5-fold if compared with the controls. The results presented here demonstrate that the expression of the new molecules described, comprising two Ig-like domains, is upregulated during the process of autograft fusion. Received: 17 November 1998 / Revised: 15 March 1999     

Genomic organization of the extracellular coding region of the human FGFR4 and FLT4 genes: Evolution of the genes encoding receptor tyrosine kinases with immunoglobulin-like domains

Receptor tyrosine kinases with five, seven, and three Ig-like domains in their extracellular region are grouped in subclasses IIIa, IIIb, and IIIc, respectively. Here, we describe the genomic organization of the extracellular coding region of the human FGFR4 (IIIc) and FLT4 (IIIb) genes and compare it to that of the human FGFR1(IIIc), KIT, and FMS (IIIa). The results show that while genes belonging to the same subclass have an identical exon/intron structure in their extracellular coding region—as they do in their intracellular coding region—genes of related subclasses only have a similar exon/intron structure. These results strongly support the hypothesis that the genes of the three subclasses evolved from a common ancestor by duplications involving entire genes, already in pieces. Hypotheses on the origin of introns and on the difference in the number of extracellular Ig-like domains in the three gene subclasses are discussed. Received: 19 August 1996 / Accepted: 2 January 1997     

Gene arrangement of the killer cell inhibitory receptor family on human chromosome 19q13.4 detected by fiber-FISH

 Human chromosome 19q13.4 has recently been revealed to be a remarkable region harboring multiple receptor genes of the immunoglobulin (Ig) superfamily differentially expressed on hematopoietic cell lineages. Over the past few years, more than 50 cDNAs have been cloned for the natural killer cell inhibitory receptor (KIR) gene family, which possess two or three Ig-like domains in the extracellular region. In this study, using two genomic DNA probes containing intron sequences of genes corresponding to the two- and three-domain types, we applied two-color-fluorescence in situ hybridization on stretched DNA fiber preparations (fiber-FISH). As a result, 11 positions homologous to KIR genes were found as a cluster within a range of approximately 120 kilobases on a chromatin fiber from human chromosome 19. Received: 7 January 1998 / Revised: 25 February 1998     

Comparative sequence analysis of bacterial symbionts from the marine sponges Geodia cydonium and Ircinia muscarum

Marine sponges (Porifera) live in a symbiotic relationship with microorganisms, primarily bacteria. Recently, several studies indicated that sponges are the most prolific source of biologically-active compounds produced by symbiotic microorganisms rather than by the sponges themselves. In the present study we characterized the bacterial symbionts from two Demospongiae, Ircinia muscarum and Geodia cydonium. We amplified 16S rRNA by PCR, using specific bacterial-primers. The phylogenetic analysis revealed the presence of nine bacterial clones from I. muscarum and ten from G. cydonium. In particular, I. muscarum resulted enriched in Bacillus species and G. cydonium in Proteobacterium species. Since these bacteria were able to produce secondary metabolites with potential biotechnological and biopharmaceutical applications, we hypothesized that I. muscarum and G. cydonium could be a considered as a “gold mine” of natural products.     

Structural Evidence for the Evolution of Pyrogenic Toxin Superantigens

Cells from metazoan organisms are eliminated in a variety of physiological and pathophysiological processes by apoptosis. In this report, we describe the cloning and characterization of molecules from the marine sponges Geodia cydonium and Suberites domuncula, whose domains show a high similarity to those that are found in molecules of the vertebrate Bcl-2 superfamily and of the death receptors. The Bcl-2 proteins contain up to four Bcl-2 homology regions (BH). Two Bcl-2-related molecules have been identified from sponges that are provided with two of those regions, BH1 and BH2, and are termed Bcl-2 homology proteins (BHP). The G. cydonium molecule, BHP1_GC, has a putative size of 28,164, while the related sequence from S. domuncula, BHP1_SD, has a M _r of 24,187. Phylogenetic analyses of the entire two sponge BHPs revealed a high similarity to members of the mammalian Bcl-2 superfamilies and to the Caenorhabditis elegans Ced-9. When the two domains, BH1 and BH2, are analyzed separately, again the highest similarity was found to the members of the Bcl-2 superfamily, but a clearly lower relationship to the C. elegans BH1 and BH2 domains in Ced-9. In unrooted phylogenetic trees the sponge BH1 and BH2 are grouped among the mammalian sequences and are only distantly related to the C. elegans BH domains. The analysis of the gene structure of the G. cydonium BHP showed that the single intron present is located within the BH2 domain at the same position as in C. elegans and rat Bcl-x_L. In addition, a sponge molecule comprising two death domains has been characterized from G. cydonium. The two death domains of the potential proapoptotic molecule GC_DD2, M _r 24,970, share a high similarity with the Fas-FADD/MORT1 domains. A death domain-containing molecule has not been identified in the C. elegans genome. The phylogenetic analysis revealed that the sponge domain originated from an ankyrin building block from which the mammalian Fas-FADD/MORT1 evolved. It is suggested that the apoptotic pathways that involve members of the Bcl-2 superfamily and of the death receptors are already present in the lowest metazoan phylum, the Porifera. Received: 27 July 1999 / Accepted: 28 December 1999     

Polymorphism and structural variation of rps16 group-II intron in the Solanum species

Nucleotide sequences of the self-splicing group-II intron of rps16 have first been determined in nine species of the Solanum genus. It was found that the observed variations in the intron length (855–864 bp) was associated with indels of 1 to 9 bp. Altogether, five indels and 50 nucleotide substitutions were detected, which were used to identify six Solanum haplotypes. Although the intron sequence was in general fairly well conserved, the distribution of the described mutations among its structural elements corresponding to six pre-RNA domains was qualitatively and quantitatively nonuniform. The highest polymorphism levels were observed in domains I, II, and IV. The sequence of domain V was absolutely invariable, which is in agreement with its functional significance. The chloroplast rpS16 intron sequences have been characterized in nine Solanum species. The intron length ranged from 855 bp to 864 bp, which is associated with 1–9-nucleotide indels. In total five indels and 50 nucleotide substitutions have been detected and six Solanum haplotypes have been revealed. Solanum rpS16 introns has been characterized by mutation rate heterogeneity between structure regions of all six domains its pre-RNA. Intron domains I, II, IV are shown to be more variable. Sequences of the domain V are invariant, that agrees with its functional significance.     

Bioinformatics analysis of plant orthologous introns: identification of an intronic tRNA-like sequence

Orthologous introns have identical positions relative to the coding sequence in orthologous genes of different species. By analyzing the complete genomes of five plants we generated a database of 40,512 orthologous intron groups of dicotyledonous plants, 28,519 orthologous intron groups of angiosperms, and 15,726 of land plants (moss and angiosperms). Multiple sequence alignments of each orthologous intron group were obtained using the Mafft algorithm. The number of conserved regions in plant introns appeared to be hundreds of times fewer than that in mammals or vertebrates. Approximately three quarters of conserved intronic regions among angiosperms and dicots, in particular, correspond to alternatively-spliced exonic sequences. We registered only a handful of conserved intronic ncRNAs of flowering plants. However, the most evolutionarily conserved intronic region, which is ubiquitous for all plants examined in this study, including moss, possessed multiple structural features of tRNAs, which caused us to classify it as a putative tRNA-like ncRNA. Intronic sequences encoding tRNA-like structures are not unique to plants. Bioinformatics examination of the presence of tRNA inside introns revealed an unusually long-term association of four glycine tRNAs inside the Vac14 gene of fish, amniotes, and mammals.     

Domain organization and intron positions inCaenorhabditis elegans collagen genes: The 54-bp module hypothesis revisited

Summary The amino acid (aa) sequences of the polypeptides encoded by five collagen genes of the nematodeCaenorhabditis elegans, col-6, col-7 (partial),col-8, col-14, andcol-19, were determined. These collagen polypeptides, as well as those encoded by the previously sequencedC. elegans collagen genescol-1 andcol-2, share a common organization into five domains: an amino-terminal leader, a short (30–33 aa) (Gly-X-Y) _n domain, a non(Gly-X-Y) spacer, a long (127–132 aa) (Gly-X-Y) _n domain, and a short carboyl-terminal domain. The domain organizations and intron positions of these polypeptides were compared with those of the polypeptides encoded byDrosophila andStrongylocentrotus type IV, and vertebrate types I, II, III, IV, and IX collagen genes; theC. elegans collagen polypeptides are most similar to the vertebrate type IX collagents. It is suggested that the collagen gene family comprises two divergent subfamilies, one of which includes the vertebrate interstitial collagen genes, and the other of which includes the invertebrate collagen genes and the vertebrate type IV and type IX collagen genes. Only the vertebrate interstitial collagen genes display clear evidence of evolution via the tandem duplication of a 54-bp exon.     

Cloning of Hsp70 genes from the marine sponges Sycon raphanus (Calcarea) and Rhabdocalyptus dawsoni (Hexactinellida). An approach to solve the phylogeny of sponges

The phylogenetic relationships among the three classes of the Porifera-Demospongiae, Calcarea and Hexactinellida-are still unresolved, despite the use of molecular analyses of rRNA. To determine whether phylogenetic resolution of these classes is possible based on genes coding for specific proteins, in the present study the genes for the 70 kDa heat shock protein [Hsp70] were isolated from Rhabdocalyptus dawsoni [Hexactinellida] and from Sycon raphanus [Calcarea], and compared to that previously isolated from the demosponge Geodia cydonium. The gene from R. dawsoni is 2021 bp long and encodes a predicted Hsp70 of Mr 77, 697; the protein comprises the characteristic sites of eukaryotic, cytoplasmic Hsp70 polypeptides. The Hsp70 isolated from cDNA from S. raphanus is 2326 bp long. It encodes a potential polypeptide of Mr 85, 927 and belongs to the same class of Hsp70s. All three sponge sequences for Hsp70 were found to be highly identical to both human and plant Hsp70s. The degree of identity at the amino acid (aa) level between the sponge sequences and the human sequence for Hsp70 is 77%-84% and at the nucleotide (nt) level, between 69% and 75%. Resolution of the phylogenetic relationship between the three classes of sponges based on the Hsp70 was not possible due to the high degree of identity [similarity] of their respective aa sequences, which ranged from 80% [90%] to 82% [91%]. The evolutionary rates-K_aa-values-calculated for the sponge Hsp70 molecules, are low, reflecting the strong functional contraints placed upon these polypeptides. These values range from 0.125 times 10^-9 for G. cydonium and R. dawsoni to 0.087 times 10^-9 for S. raphanus. Higher values have previously been reported for the G. cydonium galectin molecule [K_aa-value of 1.7 times 10^-9] and the receptor tyrosine kinase [1.24 times 10^-9] from the same animal. The occurrence of at least one double mutation, in the codon for the aa Ser in the conserved regions of the Hsp70 sequences, also suggests that these molecules are subjected to strong functional constraints.     

Characterization of a prion protein (PrP) gene from rabbit; a species with apparent resistance to infection by prions

The prion protein gene (PrP) encodes a cellular protein of unknown function. A conformational isoform of this protein is involved in the neurodegenerative prion diseases. To facilitate the identification of structurally and antigenically important regions within the PrP molecule, the rabbit PrP open reading frame (ORF) was cloned and characterised. There is 82–87% identity at the nucleotide sequence level and 88–93% identity at the amino acid (aa) sequence level, between the rabbit gene and PrP sequences of other mammals. The rabbit gene shares structural and organisational features common to all known PrP genes signifying that it is the rabbit PrP gene. Comparison of the rabbit PrP aa sequence with PrP aa sequences from different species revealed several potential epitopes. Two anti-ovine PrP peptide Ab raised in rabbits, 168-92 and 98-92, confirmed that two separate cross-reacting epitopes segregate with single aa differences between rabbit and sheep PrP at positions 43 and 99 of the rabbit PrP polypeptide. The presence of these epitopes correlates with the species recognition patterns of previously published Ab. The usefulness of the rabbit PrP gene sequence in predicting antigenic regions within the PrP proteins of various species is illustrated. The structure of the rabbit PrP protein in relation to rabbits' apparent resistance to infection by prions is discussed.     

Cloning and characterization of the actin-encoding gene of Chlamydomonas reinhardtii

The genomic and complementary DNA sequences were determined for the unique actin-encoding gene in Chlamydomonas reinhardtii (Cr). The deduced amino acid (aa) sequence of this actin was similar to most known actin sequences, with the highest identity (98.1%) being with that of Volvox carteri actin. The Cr actin-encoding gene has one intron in the 5′-untranslated region and eight introns in the coding region. The latter eight introns occur at the same positions as those in the V. carteri actin-encoding gene. The 5′-upstream region contains four short stretches of sequence similar to the so-called ‘tub box’, a characteristic sequence proposed to be responsible for the regulation of synthesis of various axonemal proteins upon deflagellation and during the cell cycle. Southern blot analysis indicated that the Cr genome has only a single actin-encoding gene. An antibody specific for the 11-aa peptide corresponding to the N-terminal sequence of this actin was found to react with a 43-kDa protein associated with flagellar inner-arm dynein. These findings indicate that a single actin functions in both the cytoplasm and flagella of this organism.     

Molecular cloning of Quek 1 and 2, two quail vascular endothelial growth factor (VEGF) receptor-like molecules

We have previously reported the cloning of two partial cDNAs corresponding to two quail (Coturnix coturnix japonica) receptor tyrosine kinases (RTKs), named Quek 1 and Quek 2, and their expression in endothelial cells of the early avian embryo. We here report the cloning of the full-size cDNAs for both molecules. Sequence comparison shows that Quek 1 and 2 share an overall amino acid (aa) identity of 49%. They both comprise seven extracellular immunoglobulin-like (Ig-like) domains, a single transmembrane domain, and an intracellular kinase domain split into two by a 70 aa insertion. These structural characteristics are shared by the members of the recently discovered VEGF receptor (VEGFR) family. We have compared the sequences of Quek 1 and 2 to the other VEGFRs. At the aa level, Quek 1 is most closely related to KDR/flk-1 (VEGFR 2) (aa identity of 69% and 71%, respectively). Quek 2 shows a similar degree of aa identity to flt-4 (VEGFR 3). Quek 1 and 2 display a lower homology to flt-1 (VEGFR 1) (about 45% aa identity). These data suggest that Quek 1 and 2 are the avian homologues of VEGFRs 2 and 3, respectively.     

S-type lectins occur also in invertebrates: High conservation of the carbohydrate recognition domain in the lectin genes from the marine sponge Geodia cydonium

The marine sponge Geodia cydonium contains several lectins.The main component, called lectin-1, is composed of three tofour identical subunits. The subunits of the lectins were clonedfrom a cDNA library; two clones were obtained. From the deducedaa sequence of one clone, LECT-1, a mol. wt of 15 313 Da iscalculated; this value is in good agreement with mass spectrometricanalysis of 15 453 25 Da. The sequence of another clone, LECT-2,was analysed and the aa sequence was deduced (15 433 Da). Thetwo subunits have a framework sequence of 38 conserved aa whichare characteristic for the carbohydrate-binding site of vertebrateS-type lectins. Clustering of lectin sequences of various speciesfollowing their pairwise comparison establishes a dendrogram,which reveals that the sponge lectin could be considered asthe ancestor for vertebrate S-type lectins. Geodia cydonium lectin sponges S-type lectin     

Structure and domain organization of the CD19 antigen of human, mouse, and guinea pig B lymphocytes. Conservation of the extensive cytoplasmic domain.

The CD19 molecule is a 95,000 Mr cell-surface protein of human B lymphocytes with two extracellular Ig-like domains and a 240 amino acid cytoplasmic tail. cDNA encoding human CD19 and the cytoplasmic domain of the mouse CD19 Ag were previously isolated. In this report, those cDNA were used to isolate cDNA or genomic DNA encoding the complete mCD19 protein and a portion of CD19 from the guinea pig. Mouse pre-B and B cell lines expressed two CD19 mRNA species of 2.7 and 2.2 kb, whereas myeloma cell lines were negative as were T cell lines. Similarly, among mouse organs, only spleen contained detectable CD19 mRNA. These results suggest that only B cells express CD19 in mouse, as in man. Sequence determination revealed substantial conservation, with hCD19 and mCD19 being 66% and hCD19 and gpCD19 being 73% identical in amino acid sequence. The cytoplasmic region of CD19 was most highly conserved with human/mouse being 73% identical and human/guinea pig being 83% identical in amino acid sequence. Isolation of the hCD19 and mCD19 genes and determination of exon/intron boundaries revealed that both genes were structurally similar and were composed of at least 15 exons, 4 encoded extracellular domains, and 9 encoded cytoplasmic domains. Six of the exons that encoded cytoplasmic domains were essentially identical in sequence in all three species indicating that these regions have undergone considerable selective pressure to conserve sequences. Thus, CD19 appears to be well conserved in structure and expression through recent mammalian evolution and the highly conserved cytoplasmic domains may play a critical role in the transduction of CD19-mediated signals.     

Molecular Evolution of TEPP Protein Genes in Metazoans

TEPP is a gene expressed in human reproductive organs such as testis, prostate, and placenta. Here, identification and molecular evolutionary analysis of TEPP proteins in various metazoan animals including deuterostomes (chordates, hemichordates, and echinoderms), lophotrochozoans (mollusks and annelids), and cnidarians (sea anemone and coral) are reported. A multiple sequence alignment revealed two highly conserved regions in TEPP proteins that had no similarity to any other known domains or proteins. Genomic sequence analysis showed frequent shifting of the splice sites of intron 1 in mammalian TEPP genes. A comparison of the intron positions in the coding region showed that the exon/intron structure of the TEPP gene was established in an early metazoan ancestor and that independent loss of a single intron occurred in echinoderms and in vertebrates. The urochordate tunicate TEPP genes are intronless, possibly due to replacement of the original gene by a retrogene. No homolog was detected in birds, insects, nematodes, and teleost fishes despite the extensive sequence data of these species, implying that the TEPP gene might be lost in these lineages.     

Cloning and sequencing of a cDNA encoding bovine intercellular adhesion molecule 3 (ICAM-3)

A cDNA encoding a putative bovine intercellular adhesion molecule (ICAM)-3, a ligand of the leukocyte integrin LFA-1 (CD11a/CD18), was sequenced and compared with human ICAM sequences. The 1635-bp bovine sequence codes for a protein of 544 amino acids (aa). This putative bovine ICAM-3 has five immunoglobulin (Ig)-like domains similar to human ICAM-1 and ICAM-3, and belongs to the Ig gene superfamily. The overall identities of the deduced aa sequence with those of human ICAM-3 and ICAM-1 are 61% and 58%, respectively. The predicted number and positions of Cys residues are all conserved between the bovine and human ICAM 3 aa sequences.     

Identification of Four Genes Coding for Isoforms of Murinoglobulin, the Monomeric Mouse α2-Macroglobulin: Characterization of the Exons Coding for the Bait Region

Murinoglobulins are the single chain members of the α₂-macroglobulin family of proteinase inhibitors in the mouse. DNA clones representing the genes coding for four different murinoglobulins were isolated from three independent mouse genomic DNA libraries. Sequence analysis demonstrated that in each gene two exons are coding for the bait region. This is the specific protein sequence in each α-macroglobulin, which is functionally important since it is extremely sensitive to cleavage by different proteinases. The molecular data established the existence of at least four different murinoglobulin genes. Three of these corresponded to the three cDNA clones previously identified. Sequencing of intron-exon boundaries and intron sizing allowed us to construct physical maps of the region from exon 15 to exon 25 (numbered in comparison to mouse α₂-macroglobulin) in each murinoglobulin gene. Southern blotting of genomic DNA from five different mouse strains confirmed this analysis and even suggested the possible existence of a fifth murinoglobulin gene. These data indicate that the mouse presents genetic repertoire of the α₂-macroglobulin family much more complex than originally anticipated. The bait region exon sequences showed a considerably higher degree of divergence (72 to 88% sequence identity) than that of the flanking exon sequences coding for adjacent, structural domains of the murinoglobulin proteinase inhibitors (91 to 96%). Even more surprising was that adjacent intron sequences are conserved as faithfully as the nonbait region coding exons (90 to 96%). These data demonstrate a unique property of the bait region coding sequences, as they apparently are allowed to mutate considerably. This divergency must then confer divergent proteinase inhibitory properties to the resulting proteins.