Merike Kelve

Origin of the interferon‐inducible (2′‐5′)oligoadenylate synthetases: cloning of the (2′‐5′)oligoadenylate synthetase from the marine sponge Geodia cydonium

In vertebrates cytokines mediate innate (natural) immunity and protect them against viral infections. The cytokine interferon causes the induction of the (2′‐5′)oligoadenylate synthetase [(2‐5)A synthetase], whose product, (2′‐5′)oligoadenylate, activates the endoribonuclease L which in turn degrades (viral) RNA. Three isoforms of (2‐5)A synthetases exist, form I (40–46 kDa), form II (69 kDa), and form III (100 kDa). Until now (2‐5)A synthetases have only been cloned from birds and mammals. Here we describe the cloning of the first putative invertebrate (2‐5)A synthetase from the marine sponge Geodia cydonium. The deduced amino acid sequence shows signatures characteristic for (2‐5)A synthetases of form I. Phylogenetic analysis of the putative sponge (2‐5)A synthetase indicates that it diverged first from a common ancestor of the hitherto known members of (vertebrate) (2‐5)A synthetases I, (2‐5)A synthetases II and III. Moreover, it is suggested that the (2‐5)A synthetases II and III evolved from this common ancestor (very likely) by gene duplication. Together with earlier results on the existence of the (2′‐5′)oligoadenylates in G. cydonium, the data presented here demonstrate that also invertebrates, here sponges, are provided with the (2‐5)A system. At present, it is assumed that this system might be involved in growth control, including control of apoptosis, and acquired its additional function in innate immune response in evolutionarily younger animals, in vertebrates.

Qualitative and quantitative aspects of 2-5A synthesizing capacity of different marine sponges

2-5A synthetase is an important component of the mammalian antiviral 2-5A system. At present, the existence of 2-5A synthetase in the lowest animals, the marine sponges, has been demonstrated, although this enzyme has not been found in bacteria, yeast or plants. Here, we studied the 2-5A synthesizing capacity and the product profile of a variety of marine sponges belonging to Demospongia subclasses Tetractinomorpha and Ceractinomorpha. The 2-5A synthetase activity varied largely, in the range of four orders of magnitude, depending on the sponge species. Compared with the enzymes of the mammalian 2-5A synthetase family, the most active sponge species exhibited a surprisingly high 2-5A synthetase specific activity. Unlike the mammalian 2-5A synthetases that produce 2-5A oligomers in the presence of a double-stranded RNA activator, the 2-5A synthetase(s) from sponges were active without the addition of dsRNA. The sponge species differed in their product profiles. A novel product pool formed by Chondrosia reniformis was identified as a series of long 2-5A oligomers (up to 17-mers) with the prevalence of heptamers and octamers. The large variability of qualitative and quantitative characteristics of sponge 2-5A synthetases may refer to the occurrence of a variety of 2-5A synthetase isozymes in sponges.

Natural occurrence of 2',5'-linked heteronucleotides in marine sponges.

2′,5′-oligoadenylate synthetases (OAS) as a component of mammalian interferon-induced antiviral enzymatic system catalyze the oligomerization of cellular ATP into 2′,5′-linked oligoadenylates (2-5A). Though vertebrate OASs have been characterized as 2′-nucleotidyl transferases under in vitro conditions, the natural occurrence of 2′,5′-oligonucleotides other than 2-5A has never been demonstrated. Here we have demonstrated that OASs from the marine sponges Thenea muricata and Chondrilla nucula are able to catalyze in vivo synthesis of 2-5A as well as the synthesis of a series 2′,5′-linked heteronucleotides which accompanied high levels of 2′,5′-diadenylates. In dephosphorylated perchloric acid extracts of the sponges, these heteronucleotides were identified as A2′p5′G, A2′ p5′U, A2′p5′C, G2′p5′A and G2′ p5′U. The natural occurrence of 2′-adenylated NAD+ was also detected. In vitro assays demonstrated that besides ATP, GTP was a good substrate for the sponge OAS, especially for OAS from C. nucula. Pyrimidine nucleotides UTP and CTP were also used as substrates for oligomerization, giving 2′,5′-linked homo-oligomers. These data refer to the substrate specificity of sponge OASs that is remarkably different from that of vertebrate OASs. Further studies of OASs from sponges may help to elucidate evolutionary and functional aspects of OASs as proteins of the nucleotidyltransferase family.

Molecular identification, characterization and distribution of freshwater sponges (Porifera: Spongillidae) in Estonia

This study is the first comprehensive survey of the distribution of freshwater sponges in the rivers of Estonia. The classical morphological method for the identification of freshwater sponges was complemented by a molecular approach, based on sequencing of a DNA region containing a variable D3 domain of the 28S rDNA. This particular segment of 28S RNA proved to be suitable for the discrimination between freshwater sponges at the species level. We showed that Ephydatia fluviatilis was the most widely distributed species followed by Spongilla lacustris and Ephydatia muelleri. Eunapius fragilis was found in a small number of localities and has been described for the first time in Estonia.

Pyrimidinoceptor potentiation by ATP in NG108-15 cells

Regulation of inositol phospholipid hydrolysis by UTP and UDP in neuroblastoma×glioma hybrid cell line NG108‐15 was potentiated in the presence of ATP. The effect of ATP was dose dependent and shifted the EC50 value for these uracil nucleotides up to three powers of magnitude, having no influence on the maximal value of the response. Adenine nucleotides (ADP, AMP, adenosine 5′‐O‐(3‐thiotriphosphate) (ATPγS), β,γ‐methyleneadenosine 5′‐triphosphate (βγMeATP), 3′‐O‐(4‐benzoyl)benzoyl ATP (BzATP) and 3′‐deoxyadenosine 5′‐O‐(1‐thio)triphosphate (dATPαS)) as well as adenosine, had no influence on the pyrimidinoceptor response. The potentiation effect was abolished by excess of EDTA. The results were in agreement with the hypothesis of pyrimidinoceptor affinity regulation via extracellular phosphorylation of the receptor protein, initiated by ATP. This mechanism may have physiological implication for functioning of uracil nucleotides as endogenous signaling molecules.

Intragenomic Profiling Using Multicopy Genes: The rDNA Internal Transcribed Spacer Sequences of the Freshwater Sponge Ephydatia fluviatilis.

Multicopy genes, like ribosomal RNA genes (rDNA), are widely used to describe and distinguish individuals. Despite concerted evolution that homogenizes a large number of rDNA gene copies, the presence of different gene variants within a genome has been reported. Characterization of an organism by defining every single variant of tens to thousands of rDNA repeat units present in a eukaryotic genome would be quite unreasonable. Here we provide an alternative approach for the characterization of a set of internal transcribed spacer sequences found within every rDNA repeat unit by implementing direct sequencing methodology. The prominent allelic variants and their relative amounts characterizing an individual can be described by a single sequencing electropherogram of the mixed amplicon containing the variants present within the genome. We propose a method for rational analysis of heterogeneity of multicopy genes by compiling a profile based on quantification of different sequence variants of the internal transcribed spacers of the freshwater sponge Ephydatia fluviatilis as an example. In addition to using conventional substitution analysis, we have developed a mathematical method, the proportion model method, to quantify the relative amounts of allelic variants of different length using data from direct sequencing of the heterogeneous amplicon. This method is based on determining the expected signal intensity values (corresponding to peak heights from the sequencing electropherogram) by sequencing clones from the same or highly similar amplicon and comparing hypothesized combinations against the values obtained by direct sequencing of the heterogeneous amplicon. This method allowed to differentiate between all specimens analysed.

THE EFFECTS OF 2-5A ON PROTEIN SYNTHESIS IN WHEAT GERM EXTRACTS AND TOBACCO PROTOPLASTS

Abstract Nonphosphorylated 2-5A inhibited translation and caused RNA degradation in wheat germ extract, whereas 3-5A had no effect. Protein synthesis inhibition by 2-5A was observed in tobacco protoplasts. 70 kD 2-5A-binding protein was found in potato leaf extracts by chemical crosslinking.

Evolutionary distribution of deoxynucleoside 5-monophosphate N-glycosidase, DNPH1

Deoxynucleoside 5-monophosphate N-glycosidase, DNPH1 is a member of the nucleoside 2-deoxyribosyltransferase (NDT) family. This enzyme catalyzes the hydrolysis of deoxynucleoside monophosphates into free nucleobase moieties and 2-deoxyribose 5-phosphates. The DNPH1 enzymatic activity was first demonstrated in rats and then in humans. Subsequently the DNPH1 gene was identified in a variety of organisms, mainly in Metazoa. Herein, we demonstrate that despite DNPH1 genes being distributed in almost all metazoans, the occurrence of DNPH1genes is mosaic. For example, they cannot be found anywhere in the entire clade of Sauropsida or anywhere in the whole phyla of Arthropoda and Ctenophora. Even among mammals, there are organisms without functional DNPH1 protein (Camelidae and most likely Cetacea). By our knowledge, the DNPH1 gene is missing in plants, fungi and in majority of protists. Accordingly, the enzyme is apparently not of vital importance in all the branches of the Tree of Life. Surprisingly the DNPH1 gene may be found in archaea as well as in bacteria. This refers to the origin of the gene from the period before the archaea branched off from other bacteria. We show that the genomic and protein primary structures of DNPH1 are highly conserved and any modification in such a structure would result in conversion to a pseudogene, which could possibly be eliminated from the genome.