Positive selection in prion protein.

The prion diseases, such as Creutzfeldt-Jakob disease of humans and bovine spongiform encephalopathy, involve the aberrant metabolism and accumulation of prion protein PrP. There are three contradictory hypotheses about evolution of prion protein gene PRNP. Population genetic studies have proposed that PRNP could be under balancing selection, strong purifying selection, or mainly positive selection. We made use of the maximum likelihood tests for detection of positive selection at the amino acid level and present availability of PRNP coding sequences to contribute to these disagreements. Positive selection could occur at amino acids residing in active sites, and at amino acids involved in protein-protein interactions. Thus we tested a hypothesis that positive selection at the amino acid level in PrP might have taken place in human and related species from the superordinal group Euarchonta, as well as in bovine and related species from the superordinal clade Laurasiatheria. Our study and the present experimental evidences indicate that positive selection at the amino acid level might have taken place in the PrP signal sequences and conformationally plastic PrP regions, as well as at the protein X binding sites.

Mitochondrial genome of Suberites domuncula: palindromes and inverted repeats are abundant in non-coding regions.

The 26,300-nucleotide sequence of the mitochondrial DNA (mtDNA) molecule of the demosponge Suberites domuncula (Olivi, 1792), the largest in size yet found in Porifera, has been determined. We describe the second hadromerid sponge mitochondrial genome that contains the same set of 41 genes as the hadromerid sponge Tethya actinia, including trnMe(cau), trnI2(cau), trnR2(ucu), and atp9, all of which are transcribed in the same direction. Furthermore, rRNA genes for the small and large ribosomal subunit are very long, rns is indeed the longest among Metazoa (1833 bp). Intergenic regions (IGR) comprise about 25% of S. domuncula mtDNA and include numerous direct and inverted repeats, as well as palindromic sequences. No overlapping genes and introns were found. Phylogenetic analyses based on concatenated amino acid sequences from twelve mitochondrial protein genes strongly support the affiliation of S. domuncula to the order Hadromerida. Moreover, we have analyzed and compared two segments of mtDNA which include the three IGR from S. domuncula (12 and 16 specimens for segments I and II) and Suberites ficus (10 and 5 for segments I and II, respectively). S. ficus has frequently been reported as being both synonymous with, as well as a separate species from S. domuncula. We have found polymorphisms in IGR of both species and long deletions (43 and 167 bp in size) in two IGR of S. ficus.

Comparative genomic analysis of prion genes.

BACKGROUND: The homologues of human disease genes are expected to contribute to better understanding of physiological and pathogenic processes. We made use of the present availability of vertebrate genomic sequences, and we have conducted the most comprehensive comparative genomic analysis of the prion protein gene PRNP and its homologues, shadow of prion protein gene SPRN and doppel gene PRND, and prion testis-specific gene PRNT so far. RESULTS: While the SPRN and PRNP homologues are present in all vertebrates, PRND is known in tetrapods, and PRNT is present in primates. PRNT could be viewed as a TE-associated gene. Using human as the base sequence for genomic sequence comparisons (VISTA), we annotated numerous potential cis-elements. The conserved regions in SPRNs harbour the potential Sp1 sites in promoters (mammals, birds), C-rich intron splicing enhancers and PTB intron splicing silencers in introns (mammals, birds), and hsa-miR-34a sites in 3'-UTRs (eutherians). We showed the conserved PRNP upstream regions, which may be potential enhancers or silencers (primates, dog). In the PRNP 3'-UTRs, there are conserved cytoplasmic polyadenylation element sites (mammals, birds). The PRND core promoters include highly conserved CCAAT, CArG and TATA boxes (mammals). We deduced 42 new protein primary structures, and performed the first phylogenetic analysis of all vertebrate prion genes. Using the protein alignment which included 122 sequences, we constructed the neighbour-joining tree which showed four major clusters, including shadoos, shadoo2s and prion protein-likes (cluster 1), fish prion proteins (cluster 2), tetrapode prion proteins (cluster 3) and doppels (cluster 4). We showed that the entire prion protein conformationally plastic region is well conserved between eutherian prion proteins and shadoos (18-25% identity and 28-34% similarity), and there could be a potential structural compatibility between shadoos and the left-handed parallel beta-helical fold. CONCLUSION: It is likely that the conserved genomic elements identified in this analysis represent bona fide cis-elements. However, this idea needs to be confirmed by functional assays in transgenic systems.

The complete set of ribosomal proteins from the marine sponge Suberites domuncula.

The siliceous marine sponge Suberites domuncula is a member of the most ancient and simplest extant phylum of multicellular animals-Porifera, which have branched off first from the common ancestor of all Metazoa. We have determined primary structures of 79 ribosomal proteins (r-proteins) from S. domuncula: 32 proteins from the small ribosomal subunit and 47 proteins from the large ribosomal subunit. Only L39 and L41 polypeptides (51 and 25 residues long in rat, respectively) are missing. The sponge S. domuncula is, after nematode Caenorhabditis elegans and insect Drosophila melanogaster the third representative of invertebrates with known amino acid sequences of all r-proteins. The comparison of S. domuncula r-proteins with r-proteins from D. melanogaster, C. elegans, rat, Arabidopsis thaliana and Saccharomyces cerevisiae revealed very interesting findings. The majority of the sponge r-proteins are more similar to their homologues from rat, than to those either from invertebrates C. elegans and D. melanogaster, or yeast and plant. With few exceptions, the overall sequence conservation between sponge and rat r-proteins is 80% or higher. The phylogenetic tree of concatenated r-proteins from 6 eukaryotic species (rooted with archaeal r-proteins) has the shortest branches connecting sponge and rat. Both model invertebrate organisms experienced recently accelerated evolution and therefore sponge r-proteins very probably better reflect structures of proteins in the ancestral metazoan ribosome, which changed only little during metazoan evolution. Furthermore, r-proteins from the plant A. thaliana are significantly closer to metazoan r-proteins than are those from the yeast S. cerevisiae.

Bauplan of urmetazoa: basis for genetic complexity of metazoa.

Sponges were first grouped to the animal-plants or plant-animals then to the Zoophyta or Mesozoa and finally to the Parazoa. Only after the application of molecular biological techniques was it possible to place the Porifera monophyletically with the other metazoan phyla, justifying a unification of all multicellular animals to only one kingdom, the Metazoa. The first strong support came from the discovery that cell-cell and cell-matrix adhesion molecules that were cloned from sponges and were subsequently expressed share a high DNA sequence and protein function similarity with the corresponding molecules of other metazoans. Besides these evolutionary novelties for Metazoa, sponges also have morphogens and transcription factors in common with other metazoan phyla. Surprisingly, even those elements exist in Porifera, which are characteristic for pattern and axis formation. Recent studies showed that epithelial layers of sponges are sealed against the extracellular milieu through tight-junction proteins. The cell culture system from sponges, the primmorphs, was suitable for understanding morphogenetic events. Finally, stem cell marker genes were isolated, which underscored that sponge cells have the capacity to differentiate. In the relatively short period of time, approximately 200 million years, the basic pathways had to be established that allowed the transition for multicellular organisms to a colonial system through the formation of adhesion molecules; based on the development of a complex immune system and the apoptotic machinery of an integrated system, the Urmetazoa, which evolved approximately 800 million years ago, could be reached. Hence, the Bauplan of the hypothetical Urmetazoa can now be constructed according to genomic regulatory systems similar to those found in higher Metazoa. These data caused a paradigmatic change; the Porifera are complex and simple but by far not primitive.

recA gene expression in a streptomycete is mediated by the unusual C-terminus of RecA protein.

Streptomyces RecA proteins are characterized by a conserved, positively charged extension of unknown function appended at their C-termini. To investigate the function of this element, we introduced the Streptomyces rimosus recA gene and its mutant form encoding the protein with a C-terminal deletion into S. rimosus. Both transcript and protein levels were dramatically increased in the strain expressing the truncated gene compared to the strain bearing the wild-type recA, indicating involvement of the characteristic C-terminal extension in regulating the recA expression in Streptomyces. Considering that RecA acts as a major regulator of DNA damage response in bacteria, this mode of regulation is expected to have broader implications and significance that outreaches our current understanding of RecA autoregulation.

Molecular markers for germ cell differentiation in the demosponge Suberites domuncula.

Sponges (phylum Porifera) are simple metazoans for which no molecular information on gametogenesis and larval development is available. To support the current study, it was confirmed by histology that oocytes and larvae were produced by the demosponge Suberites domuncula. Three genes/expressed products from S. domuncula whose expression correlated with sexual reproduction were identified and characterized (they are used here as marker genes): i) a receptor tyrosine kinase (RTK) with sequence similarity in the tyrosine kinase domain to fibroblast growth factor receptors; ii) the sex-determining protein FEM1 and iii) the sperm associated antigen (SAA) of triploblasts. Antibodies against the extracellular domain of the RTK specifically stained oocytes and larvae in S. domuncula tissue sections. Induction of these three genes was successful at elevated temperature, a factor which also promotes natural gametogenesis. In situ hybridization analyses revealed that FEM1 and SAA were expressed in those areas in which gametogenesis begins. Our results indicate that genes which play a role in sex determination may be present in Porifera.

Src proteins/src genes: from sponges to mammals.

The genome of marine sponge Suberites domuncula, a member of the most ancient and most simple metazoan phylum Porifera, encodes at least five genes for Src-type proteins, more than, i.e., Caenorhabditis elegans or Drosophila melanogaster (two in each). Three proteins, SRC1SD, SRC2SD and SRC3SD, were fully characterized. The overall homology (identity+similarity) among the three S. domuncula Srcs (68-71%) is much lower than the sequence conservation between orthologous Src proteins from freshwater sponges (82-85%). It is therefore very likely that several src genes/proteins were already present in the genome of Urmetazoa, the hypothetical metazoan ancestor. We have identified in the S. domuncula expressed sequence tags (ESTs) database further Src homology 2 (SH2) and 3 (SH3) domains that are unrelated to protein tyrosine kinases (PTKs). Src-related SH2 and SH3 domains from different species are much more conserved than SH2 and SH3 domains from different proteins in the same organism (S. domuncula), supporting the view that the common, ancestral src gene was already a multidomain protein composed of SH3, SH2 and tyrosine kinase (TK) domains. Two S. domuncula src genes were fully sequenced: src1SD gene has six and src2SD gene only one intron in front of SH2 domain, located at the same position in both genes. All vertebrate src genes, from fish to human, originated from the same ancestral gene, because they all have 10 introns at conserved positions. However, src genes in invertebrates have fewer introns that are located at different positions. Only the intron in front of the SH2 domain is present at the absolutely conserved position (and phase) in all known src genes, indicating that at least this intron was already present in the ancestral gene, common to all Metazoa. Our results also suggest that TK domain in this ancestral src was encoded on a single exon.

Conservation of the positions of metazoan introns from sponges to humans.

Sponges (phylum Porifera) are the phylogenetic oldest Metazoa still extant. They can be considered as reference animals (Urmetazoa) for the understanding of the evolutionary processes resulting in the creation of Metazoa in general and also for the metazoan gene organization in particular. In the marine sponge Suberites domuncula, genes encoding p38 and JNK kinases contain nine and twelve introns, respectively. Eight introns in both genes share the same positions and the identical phases. One p38 intron slipped for six bases and the JNK gene has three more introns. However, the sequences of the introns are not conserved and the introns in JNK gene are generally much longer. Introns interrupt most of the conserved kinase subdomains I-XI and are found in all three phases (0, 1 and 2). We analyzed in details p38 and JNK genes from human, Caenorhabditis elegans and Drosophila melanogaster and found in most genes introns at the positions identical to those in sponge genes. The exceptions are two p38 genes from D. melanogaster that have lost all introns in the coding sequence. The positions of 11 introns in each of four human p38 genes are fully conserved and ten introns occupy identical positions as the introns in sponge p38 or JNK genes. The same is true for nine, out of ten introns in the human JNK-1 gene. The introns in human p38 and JNK genes are on average more than ten times longer than corresponding introns in sponges. It was proposed that yeast HOG1-like kinases (from i.e. Saccharomyces cerevisiae and Emericella nidulans) and metazoan p38 and JNK kinases are orthologues. p38 and JNK genes were created after the split from fungi by the duplication and diversification of the HOG1-like progenitor gene. Our results further support the common origin of p38 and JNK genes and speak in favor of a very early time of duplication. The ancestral gene contained at least ten introns, which are still present at the very conserved positions in p38 and JNK genes of extant animals. Four of these introns are present at the same positions in the HOG-like gene in the fungus E. nidulans. The others probably entered the ancestral gene after the split of fungi, but before the duplication of the gene and before the creation of the common, urmetazoan progenitor of all multicellular animals. A second gene coding for an immune molecule is described, the allograft inflammatory factor, which likewise showed a highly conserved exon/intron structure in S. domuncula and in human. These data show that the intron/exon borders are highly conserved in genes from sponges to human.

Identification of a promoter motif regulating the major DNA damage response mechanism of Mycobacterium tuberculosis.

The principal response of many bacteria to DNA damage is mediated by a mechanism dependent on the LexA and RecA proteins. However, Mycobacterium tuberculosis was recently reported to regulate a majority of DNA repair genes independently of RecA and LexA, suggesting that an unknown RecA/LexA-independent mechanism controls the major DNA damage response pathway in this organism. Here we have identified a motif tTGTCRgtg-8nt-TAnnnT that defines a novel RecA/LexA-independent promoter (RecA-NDp) of M. tuberculosis. Furthermore, we show that the RecA-NDp type of promoter precedes DNA repair genes in other Actinomycetales.

Transcriptional analysis of the recA gene in Streptomyces rimosus: identification of the new type of promoter.

Using primer-extension analysis we identified two transcription start sites for the recA gene in Streptomyces rimosus. A longer, weak transcript is initiated from the distal SEP promoter that contains a Cheo box like sequence: GAAC-N4-ATTC. However, the major start site of transcription is a G at position -36 and this shorter transcript significantly increases in response to DNA damage by UV-light. The -35 box (TTGTCA) and -10 box (TAGCGT) of the strong recA promoter are only 11 bp apart and this proximal promoter is almost identical to the strong, DNA damage-inducible promoter of Mycobacterium tuberculosis recA gene. We inspected the Streptomyces coelicolor database and found this type of promoter in the upstream regions of many (potentially) UV-inducible genes as well as some other genes/ORFs. Moreover, the DNA sequence between the predicted -35 and -10 boxes is also partially conserved. The consensus sequence for this new type of promoter in Streptomyces is: TTGTCAGTGGC-N6-TAGggT.

Ras-like small GTPases form a large family of proteins in the marine sponge Suberites domuncula.

Sponges (Porifera) are the simplest and the most ancient metazoan animals, which branched off first from the common ancestor of all multicellular animals. We have inspected approximately 13,000 partial cDNA sequences (ESTs) from the marine sponge Suberites domuncula and have identified full or partial cDNA sequences coding for approximately 50 different Ras-like small GTPases. Forty-four sponge proteins from the Ras family are described here: 6 proteins from the Ras subfamily, 5 from Rho, 6 from Arf, 1 Ran, and 26 Rabs or Rab-like proteins. No isoforms of these proteins were detected; the closest related proteins are two Rho proteins with 74% identity. Small GTPases from sponge display a higher degree of sequence conservation with orthologues from vertebrates (53%-93% identity) than with those from either Caenorhabditis elegans or Drosophila melanogaster. The real number of small GTPases in this sponge is certainly much higher than 50, because the actual S. domuncula database of approximately 13,000 ESTs contains at most 3000 nonredundant cDNA sequences. The number of genes for Ras-like small GTPases in yeast, C. elegans, D. melanogaster, and humans is 30, 56, 90, and 174, respectively. Both model invertebrates have only 29 Rabs or Rab-like proteins, compared with 26 already found in sponge, and are missing at least 1 Rab (Rab24) present in S. domuncula and mammals. Our results indicate that duplications and diversifications of genes encoding Ras-like small GTPases, especially the Rab subfamily of small GTPases, happened very early in the evolution of Metazoa.

Evolution of osmosensing signal transduction in Metazoa: stress-activated protein kinases p38 and JNK.

Sponges (Porifera) represent the most basal branch of the Metazoa alive today. We show that two central stress-activated protein kinases involved in the osmosensing pathway, p38 mitogen-activated protein kinase (MAPK) and JNK, can complement for the ancestral MAPK Hog1 in the yeast Saccharomyces cerevisiae. S. cerevisiae mutants lacking Hog1 (hog1-Delta 1) have been complemented with the sponge SDJNK and SDp38 genes. Western blotting has revealed that, after transformation, the hog1-Delta 1+ SDJNK(sense) and hog1-Delta 1+ SDp38(sense) clones express the sponge proteins. Functional studies have demonstrated that the complemented clones grow under hyperosmotic conditions (0.6 M NaCl). Furthermore, the expressed sponge kinases undergo phosphorylation in S. cerevisiae at 0.6 M NaCl. This report documents that the metazoan signal transduction kinases, p38 and JNK, which were originally derived from an common ancestor with yeast HOG1, have retained their function after their specification.

Evolution of metazoan cell junction proteins: the scaffold protein MAGI and the transmembrane receptor tetraspanin in the demosponge Suberites domuncula.

Until recently the positioning of the sponges (phylum Porifera) within the metazoan systematics was hampered by the lack of molecular evidence for the existence of junctional structures in the surface cell layers. In this study two genes related to the tight junctions are characterized from the demosponge Suberites domuncula: tetraspanin (SDTM4SF), a cell surface receptor, and MAGI (SDMAGI), a MAGUK (membrane-associated guanylate kinase homologue) protein. Especially the MAGI protein is known in other metazoan animal phyla to exist exclusively in tight junctions. The characteristic domains of MAGI proteins (six PDZ domains, two WW domains, and a truncated guanylate kinase motif) are conserved in the sponge protein. The functional analysis of SDMAGI done by in situ hybridization shows its expression in the surface epithelial layers (exopinacoderm and endopinacoderm). Northern blot studies reveal that expression of SDMAGI and SDTM4SF increases after formation of the pinacoderm layer in the animals as well as in primmorphs. These results support earlier notions that sponges contain junctional structures. We conclude that sponges contain epithelia whose cells are organized by cell junctions.

Emergence and disappearance of an immune molecule, an antimicrobial lectin, in basal metazoa. A tachylectin-related protein in the sponge Suberites domuncula.

Sponges (phylum Porifera) represent the evolutionarily oldest metazoans that comprise already a complex immune system and are related to the crown taxa of the protostomians and the deuterostomians. Here, we demonstrate the existence of a tachylectin-related protein in the demosponge Suberites domuncula, termed Suberites lectin. The MAPK pathway was activated in response to lipopolysaccharide treatment of the three-dimensional cell aggregates, the primmorphs; this process was abolished by the monosaccharide D-GlcNAc. The cDNA encoding the S. domuncula lectin was identified and cloned; it comprises 238 amino acids (26 kDa) in the open reading frame. The deduced protein has one potential transmembrane region, three characteristic Cys residues, and six internal tandem repeats; it shares the highest sequence similarity with lectins from the horseshoe crab Tachypleus trunculus. The steady-state level of expression of the Suberites lectin rises in primmorphs in response to lipopolysaccharide, an effect that was prevented by co-incubation with D-GlcNAc. The natural sponge lectin was purified by affinity chromatography; it has a size of 27 kDa and displays antibacterial activity against the Gram-negative bacteria Escherichia coli and the Gram-positive bacteria Staphylococcus aureus. The putative protein, deduced from the cloned gene, is identical/similar to the purified natural protein, as demonstrated by immunological cross-reactivity with specific antibodies. We conclude that the S. domuncula lectin acts as an antibacterial molecule involved in immune defense against bacterial invaders.

A (1-->3)-beta-D-glucan recognition protein from the sponge Suberites domuncula. Mediated activation of fibrinogen-like protein and epidermal growth factor gene expression.

Sponges (phylum Porifera) live in a symbiotic relationship with microorganisms, primarily bacteria. Until now, molecular proof for the capacity of sponges to recognize fungi in the surrounding aqueous milieu has not been available. Here we demonstrate, for the demosponge Suberites domuncula (Porifera, Demospongiae, Hadromerida), a cell surface receptor that recognizes (1-->3)-beta-D-glucans, e.g. curdlan or laminarin. This receptor, the (1-->3)-beta-D-glucan-binding protein, was identified and its cDNA analysed. The gene coding for the 45 kDa protein was found to be upregulated in tissue after incubation with carbohydrate. Simultaneously with the increased expression of this gene, two further genes showed an elevated steady state level of expression; one codes for a fibrinogen-like protein and the other for the epidermal growth factor precursor. Expression of the (1-->3)-beta-D-glucan-binding protein and the fibrinogen-like protein occurred in cells on the sponge surface, in the pinacoderm. By Western blotting, the product of the fibrinogen-like protein gene was identified, the recombinant protein isolated, and antibodies raised to this protein. Their application revealed that a 5 kDa factor is produced, which is apparently processed from the 77 kDa epidermal growth factor precursor. Finally, we provided evidence that a tyrosine kinase pathway is initiated in response to exposure to D-glucan; its phosphorylation activity could be blocked by aeroplysinin. In turn, the increased expression of the downstream genes was suppressed. We conclude that sponges possess a molecular mechanism for recognizing fungi via the d-glucan carbohydrates on their surfaces.