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Structure, location and expression of the Drosophila sps2 gene. (A) Genomic organization of Dsps2 showing that the gene is composed of four exons; coding sequences (black bars) and untranslated regions (open bars) are indicated. Note the position of the UGA in position 24 of the deduced protein. (B) In situ hybridization of Dsps2 cDNA to polytene chromosome showing a signal in section 31D/E (see text). (C) Northern blot showing a single transcript in poly(A) + RNA of embryos, larvae and adults; rpL9 is a control for similar RNA loading. (D–G) In situ hybridization to ovaries (D), blastoderm (E), gastrula (F) and a germ band retracted embryo (G) showing that maternal Dsps2 transcripts are expressed in nurse cells (D) and that zygotic expression occurs in a spatially restricted pattern (see text). Orientation: anterior to the left, lateral view, dorsal is top (E–G). For staging see Campos-Ortega and Hartenstein (1985).
Source publication
Synthesis of monoselenophosphate, the selenium donor required for the synthesis of selenocysteine (Sec) is catalyzed by the enzyme selenophosphate synthetase (SPS), first described in Escherichia coli. SPS homologs were identified in archaea, mammals and Drosophila. In the latter, however, an amino acid replacement is present within the catalytic d...
Contexts in source publication
Context 1
... structure of the Dsps2 gene, as revealed by sequence comparison of cDNA and corresponding genomic DNA, is shown in Figure 1A. Dsps2 contains four exons, spanning a genomic region of ∼2 kb ( Figure 1A). ...
Context 2
... structure of the Dsps2 gene, as revealed by sequence comparison of cDNA and corresponding genomic DNA, is shown in Figure 1A. Dsps2 contains four exons, spanning a genomic region of ∼2 kb ( Figure 1A). The locus is located on the left arm of the second chromosome in section 31D/E ( Figure 1B) and codes for a single transcript of ∼1.5 kb ( Figure 1C). ...
Context 3
... contains four exons, spanning a genomic region of ∼2 kb ( Figure 1A). The locus is located on the left arm of the second chromosome in section 31D/E ( Figure 1B) and codes for a single transcript of ∼1.5 kb ( Figure 1C). ...
Context 4
... contains four exons, spanning a genomic region of ∼2 kb ( Figure 1A). The locus is located on the left arm of the second chromosome in section 31D/E ( Figure 1B) and codes for a single transcript of ∼1.5 kb ( Figure 1C). ...
Context 5
... in situ hybridization of ovaries and embryos shows that Dsps2 transcripts are expressed during all stages of oogenesis in nurse cells ( Figure 1D). They accumulate in the oocyte and remain present at high levels up to the blastoderm stage ( Figure 1E). ...
Context 6
... in situ hybridization of ovaries and embryos shows that Dsps2 transcripts are expressed during all stages of oogenesis in nurse cells ( Figure 1D). They accumulate in the oocyte and remain present at high levels up to the blastoderm stage ( Figure 1E). Subsequently, the maternally derived tran- scripts decrease (not shown) and become replaced by zygotic- ally expressed transcripts first detected in the midgut anlagen ( Figure 1F). ...
Context 7
... accumulate in the oocyte and remain present at high levels up to the blastoderm stage ( Figure 1E). Subsequently, the maternally derived tran- scripts decrease (not shown) and become replaced by zygotic- ally expressed transcripts first detected in the midgut anlagen ( Figure 1F). During subsequent stages, transcripts become enriched in a variety of tissues and organs including gut and nervous system ( Figure 1G). ...
Context 8
... the maternally derived tran- scripts decrease (not shown) and become replaced by zygotic- ally expressed transcripts first detected in the midgut anlagen ( Figure 1F). During subsequent stages, transcripts become enriched in a variety of tissues and organs including gut and nervous system ( Figure 1G). ...
Context 9
... the Dsps2 3′UTR-dependent readthrough activity is reduced more than 2-fold in embryos homozygous for a Dsps2 deficiency, which derived from hetero- zygous Dsps2 deficiency females ( Figure 4B). Residual DSPS2 in these embryos can be attributed to maternal Dsps2 activity (see Figure 1D, E). In the absence of distinct Dsps2, this activity could not be removed since additional essential genes are uncovered by the smallest available Dsps2 deficiency. ...
Citations
... Humans express 25 selenoproteins, of which three are conserved in Drosophila (68)(69)(70)(71). Of the three Drosophila selenoproteins, selenophosphate synthetase 2 (SPS2) is broadly expressed and, intriguingly, is not only a selenoprotein but also performs a key role in selenocysteine biosynthesis by promoting the formation of the selenium donor, selenophosphate (72,73). Thus, loss of SPS2 impairs all selenoprotein synthesis. ...
Post-transcriptional modification of RNA regulates gene expression at multiple levels. ALKBH8 is a tRNA modifying enzyme that methylates wobble uridines in specific tRNAs to modulate translation. Through methylation of tRNA-selenocysteine, ALKBH8 promotes selenoprotein synthesis and regulates redox homeostasis. Pathogenic variants in ALKBH8 have been linked to intellectual disability disorders in the human population, but the role of ALKBH8 in the nervous system is unknown. Through in vivo studies in Drosophila , we show that ALKBH8 controls oxidative stress in the brain to restrain synaptic growth and support learning and memory. ALKBH8 null animals lack wobble uridine methylation and exhibit a global reduction in protein synthesis, including a specific decrease in selenoprotein levels. Loss of ALKBH8 or independent disruption of selenoprotein synthesis results in ectopic synapse formation. Genetic expression of antioxidant enzymes fully suppresses synaptic overgrowth in ALKBH8 null animals, confirming oxidative stress as the underlying cause of dysregulation. ALKBH8 animals also exhibit associative learning and memory impairments that are reversed by pharmacological antioxidant treatment. Together, these findings demonstrate the critical role of tRNA modification in redox homeostasis in the nervous system and reveal antioxidants as a potential therapy for ALKBH8-associated intellectual disability.
Significance Statement
tRNA modifying enzymes are emerging as important regulators of nervous system development and function due to their growing links to neurological disorders. Yet, their roles in the nervous system remain largely elusive. Through in vivo studies in Drosophila , we link tRNA methyltransferase-regulated selenoprotein synthesis to synapse development and associative memory. These findings demonstrate the key role of tRNA modifiers in redox homeostasis during nervous system development and highlight the potential therapeutic benefit of antioxidant-based therapies for cognitive disorders linked to dysregulation of tRNA modification.
... It remains to be elucidated whether these anti-and pro-tumorigenic effects are tumor stage or grade-dependent. The known selenoproteins in D. melanogaster are dSPS2, dSelK (former called dSelG) and dSelH (also known as dSelM or BthD) (Hirosawa-Takamori et al. 2000;Castellano et al. 2001). dSelK has one cysteine homolog and dSelH has two (Castellano et al. 2001;Martin-Romero et al. 2001). ...
Drosophila melanogaster is used as a model system in biomedical studies. Selenoprotein is the major biological form of selenium in eukaryotes. Selenoproteins are generally involved in catabolic pathways in bacteria and archaea, whereas it participates in anabolic and antioxidant processes in eukaryotic. In this study, anticancer potential of selenoprotein BthD of D. melanogaster was investigated using bioinformatics methods. Results showed that selenoprotein BthD of D. melanogaster may have dual properties as evident by its orthology with selenoprotein H (SelH) of Homo sapiens and conserved domain of fructokinase-like protein 2 of Vitis vinifera. These dual properties were also revealed in the phylogenetic analysis, while further structural modeling showed that selenoprotein BthD possibly exists as homotetramer in the native functional structure. The anticancer property of selenoprotein BthD was proposed to be by synergy of antioxidant or redox activities of thioredoxin and glutathione reductase (TGR) domain and the signaling function of fructokinase-like protein 2 domain both in Golgi apparatus and cytoplasm, through energy deprivation. The anticancer peptide CRSUR was identified from conserved region of selenoprotein BthD, of which its cyclic form showed potential anticancer properties predictively through E3 ubiquitin-protein ligase regulating NF-kappa-B signaling by unleashing cells for spontaneous formation of the ripoptosome.
... The use of Drosophila melanogaster in toxicological studies has increased [42][43][44][45][46][47][48][49][50][51][52] given the genome of flies has homology to the human genome [53], thus making it a highly predictive model of toxicity in vertebrates. D. melanogaster has numerous thiol-containing proteins involved in redox signaling [54] and five selenoproteins [55][56][57][58][59]. One of these selenoproteins is selenophosphate synthetase [55], which catalyzes the synthesis of monoselenophosphate. ...
... D. melanogaster has numerous thiol-containing proteins involved in redox signaling [54] and five selenoproteins [55][56][57][58][59]. One of these selenoproteins is selenophosphate synthetase [55], which catalyzes the synthesis of monoselenophosphate. The other four identified selenoproteins are glycine-rich selenoprotein (SelG) [56], selenoprotein birthday (Bthd) [57], ring canal kelch protein (Kel) [58], and glucose dehydrogenase (Gld) [59]. ...
Background:
Exposure to vinylcyclohexene (VCH) and methylmercury (MeHg+) can induce oxidative stress and gene modulation. Several studies have been evaluating the effects of VCH and MeHg+, but little is known about interactive effects between them. This work aimed to assess the exposure and co-exposure effects of MeHg+ and VCH on oxidative stress and gene modulation in Drosophila melanogaster.
Methods:
Reactive species production, glutathione S-transferase (GST) and acetylcholinesterase (AChE) activities were evaluated after exposure and co-exposure to VCH (1 mM) and MeHg+ (0.2 mM) for one or three days in the head and body (thorax and abdomen) of flies. The expression of genes related to redox state and inflammatory response was evaluated after exposure and co-exposure to VCH and MeHg+ for three days.
Results:
Survival decreased only in flies co-exposed to VCH and MeHg+ for three days. All treatments increased total reactive species production after one day of exposure. However, no significant changes were observed in the head after three days of exposure. One day of exposure to VCH caused an increase in the head GST activity, whereas MeHg+ induced an increase after three days of exposure. Regarding the body, all treatments increased GST activity after one day of exposure, but only the flies exposed to MeHg+ presented an increase in GST activity after three days of exposure. Treatments did not alter AChE activity in the head. As for gene expression, there was a significant increase in the Relish transcription factor gene in the flies' body, but Nrf2, Keap1, Jafrac1, TrxR1, and NF-κβ were not altered.
Conclusion:
The results suggest that exposure to VCH and MeHg+ induce oxidative stress and activation of an inflammatory response in fruit flies.
... In eukaryotes, selenoproteins show a mosaic occurrence, with some organisms such as vertebrates and algae having dozens of these proteins whereas other organisms such as higher plants and fungi have lost all selenoproteins during evolution (Lobanov et al., 2009). D. melanogaster preserves only three selenoproteins that are not essential for its viability and only play a role under certain stress conditions, such as starvation (Hirosawa-Takamori et al., 2000;Missirlis et al., 2003;Shchedrina et al., 2011). Moreover, D. melanogaster does not possess a SeGPx. ...
The selenium-dependent glutathione peroxidase (SeGPx) is a well-studied enzyme that detoxifies organic and hydrogen peroxides and provides cells or extracellular fluids with a key antioxidant function. The presence of a SeGPx has not been unequivocally demonstrated in insects. In the present work, we identified the gene and studied the function of a Rhodnius prolixus SeGPx (RpSeGPx). The RpSeGPx mRNA presents the UGA codon that encodes the active site selenocysteine (Sec) and a corresponding Sec insertion sequence (SECIS) in the 3' UTR region. The encoded protein includes a signal peptide, which is consistent with the high levels of GPx enzymatic activity in the insect's hemolymph, and clusters phylogenetically with the extracellular mammalian GPx03. This result contrasts with all other known insect GPxs, which use a cysteine residue instead of Sec and cluster with the mammalian phospholipid hydroperoxide GPx04. RpSeGPx is widely expressed in insect organs, with higher expression levels in the fat body. RNA interference (RNAi) was used to reduce RpSeGPx gene expression and GPx activity in the hemolymph. Adult females were apparently unaffected by RpSeGPx RNAi, whereas first instar nymphs showed a three-day delay in ecdysis. Silencing of RpSeGPx did not alter the gene expression of the antioxidant enzymes catalase, xanthine dehydrogenase and a cysteine-GPx, but it reduced the levels of the dual oxidase and NADPH oxidase 5 transcripts that encode for enzymes releasing extracellular hydrogen peroxide/superoxide. Collectively, our data suggest that RpSeGPx functions in the regulation of extracellular (hemolymph) redox homeostasis of R. prolixus.
... Selenophosphate serves as a universal selenium donor in cellular processes both in prokaryotes and eukaryotes, and its formation is catalyzed by selenophosphate synthetase (SPS), a selD gene product [23]. However, an attempt to substitute the SPS II gene from mammals for the prokaryotic selD gene was not successful: when the SPS II gene was inserted into a selD-deficient E. coli culture, the culture growth was distinctly inhibited that indicated a lethality of the bacterial gene replacement by a homolog from mammals [3]. ...
The transformation of the strain - with plasmid vector pET11a containing the cloned gene of bacterial selenophosphate synthetase (SPS), selD, from the E. coli BL21-Gold (DE3) strain gives an overproducing strain of SPS with one synonymic substitution, E197D. The transformation efficiency was estimated as 8 × 108 CFU/μg plasmid DNA. 28 mg of highly purified preparation of recombinant SPS capable of binding TNP-ATP was eluted from DEAE-Sephadex column in amount of 15 % from the total soluble protein in crude extract. The fluorescent derivative of ATP, 2′(3′)-O-(2,4,6-trinitrophenyl)adenosine-5′-triphosphate (TNP-ATP), was used as a synthetic analog of the substrate for the monitoring and quantitative analysis of the functional activity of SPS. The non-linear regression analysis of the saturation curve of TNP-ATP binding to D197 SPS with GraphPad Prism software fits to a model with 2 distinct binding sites with
different in order. The SPS existence in a form of tetramer in given reaction conditions, in accordance with the concentration stoichiometry of 4 moles of TNP-ATP to 1 mole of recombinant protein, is being discussed. The tetramer structure was predicted with molecular modelling software YASARA and modelled in vacuum using steepest descent minimization energy method. We hypothesize here the recombinant SPS exists as a dimer in solution with two active sites capable of ATP binding in each subunit.
... The fruit fly Drosophila melanogaster has 3 selenoproteins: selenophosphate synthetase 2 (dSPS2), selenoprotein H (dSelH, also known as BthD), and selenoprotein K (dSelK, also known as G-rich) (15)(16)(17)(18). The SPS2 function (synthesis of monoselenophosphate from selenide) is essential for Sec biosynthesis and expression of selenoproteins (19,20). ...
Selenoproteins are essential in vertebrates because of their crucial role in cellular redox homeostasis, but some invertebrates that lack selenoproteins have recently been identified. Genetic disruption of selenoprotein biosynthesis had no effect on lifespan and oxidative stress resistance of Drosophila melanogaster. In the current study, fruit flies with knock-out of the selenocysteine-specific elongation factor were metabolically labeled with (75)Se; they did not incorporate selenium into proteins and had the same lifespan on a chemically defined diet with or without selenium supplementation. These flies were, however, more susceptible to starvation than controls, and this effect could be ascribed to the function of selenoprotein K. We further expressed mouse methionine sulfoxide reductase B1 (MsrB1), a selenoenzyme that catalyzes the reduction of oxidized methionine residues and has protein repair function, in the whole body or the nervous system of fruit flies. This exogenous selenoprotein could only be expressed when the Drosophila selenocysteine insertion sequence element was used, whereas the corresponding mouse element did not support selenoprotein synthesis. Ectopic expression of MsrB1 in the nervous system led to an increase in the resistance against oxidative stress and starvation, but did not affect lifespan and reproduction, whereas ubiquitous MsrB1 expression had no effect. Dietary selenium did not influence lifespan of MsrB1-expressing flies. Thus, in contrast to vertebrates, fruit flies preserve only three selenoproteins, which are not essential and play a role only under certain stress conditions, thereby limiting the use of the micronutrient selenium by these organisms.
... Vertebrate genomes encode up to 25–26 selenoproteins but surprisingly larger selenoproteomes can be found in aquatic unicellular organisms (26). Only three selenoprotein genes have been discovered in Drosophila melanogaster, SPS2, SelH and SelK (27,28). SPS2 is the selenophosphate synthetase involved in selenocysteine biosynthesis. ...
Selenoproteins contain the amino acid selenocysteine which is encoded by a UGA Sec codon. Recoding UGA Sec requires a complex
mechanism, comprising the cis-acting SECIS RNA hairpin in the 3′UTR of selenoprotein mRNAs, and trans-acting factors. Among these, the SECIS Binding Protein 2 (SBP2) is central to the mechanism. SBP2 has been so far functionally
characterized only in rats and humans. In this work, we report the characterization of the Drosophila melanogaster SBP2 (dSBP2). Despite its shorter length, it retained the same selenoprotein synthesis-promoting capabilities as the mammalian
counterpart. However, a major difference resides in the SECIS recognition pattern: while human SBP2 (hSBP2) binds the distinct
form 1 and 2 SECIS RNAs with similar affinities, dSBP2 exhibits high affinity toward form 2 only. In addition, we report the
identification of a K (lysine)-rich domain in all SBP2s, essential for SECIS and 60S ribosomal subunit binding, differing
from the well-characterized L7Ae RNA-binding domain. Swapping only five amino acids between dSBP2 and hSBP2 in the K-rich
domain conferred reversed SECIS-binding properties to the proteins, thus unveiling an important sequence for form 1 binding.
... In prokaryotes, SECIS elements reside immediately downstream of the UGA, whereas in archaea and eukaryotes, the corresponding SECIS elements reside in the 3Јuntranslated regions of the selenoprotein coding mRNA (reviewed in refs. 1, 8, 10 -12). In eukaryotes, the EF-Tu-like SelB function is split between eEFsec and SECIS-binding protein 2 (SBP2) (12)(13)(14), two components that are conserved between mammals and invertebrates such as Drosophila melanogaster (15,16 Here we report on a novel component of the Drosophila UGA read-through system, an evolutionarily conserved GTPase-activating protein, termed GAPsec. To distinguish between GAPsec in Drosophila (d) and mouse (m), we refer to the homologous GAPsec proteins as dGAPsec and mGAPsec, respectively. ...
... Flies used to examine UGA read-through were described previously (15). PBac{PB}CG5978 c05919 flies were obtained from the Bloomington stock collection (http://flystocks. ...
... cDNAs were cloned into pUAST vectors (KpnI site) and used for P element transformation. For overexpression in 3T3 cells, mGAPsec, mGAPsecR129K, and meEFsec were cloned into the KpnI site of pcDNA3.1 (for details, see ref. 15). ...
Translational read-through of the UGA stop codon is an evolutionarily conserved feature that most prominently represents the basis of selenoprotein biosynthesis. It requires a specific cis-acting stem loop control element, termed SECIS, which is located in the 3'-untranslated region of eukaryotic selenoprotein mRNAs. In a search for novel factors underlying the SECIS-directed UGA read-through process, we identified an evolutionary conserved GTPase-activating protein, termed GAPsec. We show that the activity of the Drosophila GAPsec (dGAPsec) is necessary to support SECIS-dependent UGA read-through activity in flies and the mouse homolog mGAPsec in mice tissue culture cells. However, selenoprotein biosynthesis is not impaired in flies that lack dGAPsec activity. The results indicate that GAPsec is part of a novel SECIS-dependent translational read-through system that does not involve selenocysteine incorporation.
... In contrast to mammalian systems, Drosophila and Anopheles, and possibly insects in general, lack glutathione reductase (GR) and must rely solely on the Trx system to maintain levels of reduced GSH (Bauer et al., 2002; Becker et al., 2003; Missirlis et al., 2002). Components of the Trx system have been described for Drosophila (Bauer et al., 2003b; Giordano et al., 2003; Hirosawa-Takamori et al., 2000; Missirlis et al., 2001; Morozova et al., 2003; Orr et al., 2003; Pellicenapalle et al., 1997; Radyuk et al., 2001; Rodriguez et al., 2000; Salz et al., 1994) the mosquito A. gambiae (Bauer et al., 2003a), and Plasmodium (Kanzok et al., 2000; Muller et al., 1996 Muller et al., , 2001 Muller et al., , 2003). In bacteria challenged Ae. aegypti and Ar. ...
Upon encountering an object recognized as foreign, insect hemocytes aggregate in multiple layers on the surfaces of the object in a process known as encapsulation. For encapsulation to occur, hemocytes must switch from their usual nonadherent state to an adherent state, presumably by regulating the activity of adhesion proteins. Although detailed knowledge exists regarding the adhesion receptors for cells of the mammalian immune system, comparable information on adhesion molecules of insect hemocytes and their function in immune responses is extremely limited. We report here the identification of an integrin present exclusively on the surface of hemocytes in the tobacco hornworm, Manduca sexta. Monoclonal antibodies MS13 and MS34, which bind to plasmatocytes and block encapsulation, were used for immunoaffinity chromatography to isolate their corresponding hemocyte antigen, which was revealed to be the same integrin beta subunit. A cDNA for this M. sexta integrin beta1 was cloned and characterized. Integrin-beta1 mRNA was detected by Northern analysis in hemocytes and not in other tissues tested. MS13 and MS34 were demonstrated to bind to a recombinant fragment of integrin beta1 consisting of the I-like domain, consistent with their blocking of a ligand-binding site and subsequent disruption of plasmatocyte adhesion. Injection of double stranded integrin-beta1 RNA into larvae resulted in decreased integrin beta1 expression in plasmatocytes and significantly suppressed encapsulation. These results indicate that activation of ligand-binding by the hemocyte-specific integrin plays a key role in stimulating plasmatocyte adhesion leading to encapsulation.
... 3. 1 Castellano et al., EMBO reports, 2, 697-702 (2001) At the time, no selenoprotein genes, besides sps2 Hirosawa-Takamori et al., 2000), were known in Drosophila. In addition, another key enzyme of the selenoprotein biosynthesis pathway, sps1, had been described and shown to be functional . ...
... After such review of selenoprotein from the biological and computational point of view, we decided to attempt the screening of the D. melanogaster genome. As mentioned above, no selenoproteins besides SPS2 (Alsina, 1999;Hirosawa-Takamori et al., 2000) were known in this model organism at that time. The main difference between a genome sequence and a database of ESTs, is that, while in size can be similar, the latter is highly redundant. ...
AbstractAlthough the genome sequence and gene content are available for an increasing number of organisms, eukaryotic selenoproteins remain poorly characterized. In these proteins, selenium (Se) is incorporated in the form of selenocysteine(Sec), the 21st amino acid. Selenocysteine is cotranslationally inserted in response to UGA codons (a stop signal in the canonical genetic code). The alternative decoding is mediated by a stem-loop structure in the 3UTR of selenoprotein mRNAs (the SECIS element). Selenium is implicated in male infertility, cancer and heart diseases, viral expression and ageing. In addition, most selenoproteins have homologues in which Sec is replaced by cysteine (Cys).Genome biologists rely on the high-quality annotation of genomes to bridge the gap from the sequence to the biology of the organism. However, for selenoproteins, which mediate the biological functions of selenium, the dual role of the UGA codon confounds both the automatic annotation pipelines and the human curators. In consequence, selenoproteins are misannotated in the majority of genome projects. Furthermore, the finding of novel selenoprotein families remains a difficult task in the newly released genome sequences.In the last few years, we have contributed to the exhaustive description of the eukaryotic selenoproteome (set of eukaryotic selenoproteins) through the development of a number of ad hoc computational tools. Our approach is based on the capacity of predicting SECIS elements, standard genes and genes with a UGA codon in-frame in one or multiple genomes. Indeed, the comparative analysis plays an essential role because 1) SECIS sequences are conserved between close species (eg. human-mouse); and 2) sequence conservation across a UGA codon between genomes at further phylogenetic distance strongly suggests a coding function (eg. human-fugu). Our analysis of the fly, human and Takifugu and Tetraodon genomes have resulted in 9 novel selenoprotein families. Therefore, 20 distinct selenoprotein families have been described in eukaryotes to date. Most of these families are widely (but not uniformly) distributed across eukaryotes, either as true selenoproteins or Cys-homologues.The correct annotation of selenoproteins is thus providing insight into the evolution of the usage of Sec. Our data indicate a discrete evolutionary distribution of selenoprotein in eukaryotes and suggest that, contrary to the prevalent thinking of an increase in the number of selenoproteins from less to more complex genomes, Sec-containing proteins scatter all along the complexity scale. We believe that the particular distribution of each family is mediated by an ongoing process of Sec/Cys interconversion, in which contingent events could play a role as important as functional constraints. The characterization of eukaryotic selenoproteins illustrates some of the most important challenges involved in the completion of the gene annotation of genomes. Notably among them, the increasing number of exceptions to our standard theory of the eukaryotic gene and the necessity of sequencing genomes at different evolutionary distances towards such a complete annotation.
