Arginine kinase isoforms in the closest protozoan relative of metazoans

The genome of the choanoflagellate Monosiga brevicollis contains at least three genes for the phosphoryl transfer enzyme, arginine kinase (AK; EC 2.7.3.3). Bioinformatic analyses of the deduced amino acid sequences of the proteins coded for by two of these genes showed that one of these AKs is cytoplasmic (denoted AK1) while the other appears to have an N-terminal mitochondrial targeting peptide (denoted AK2). Cloning and expression of the cDNA for AK1 yielded considerable soluble AK activity. Three AK2 constructs were expressed - one corresponding to the full length protein and two corresponding to truncated versions in which the signal peptide had been deleted. Expression of the former construct yielded minimal soluble activity. In contrast, significant AK activity was found in both truncated constructs confirming the importance of removal of the targeting peptide for proper folding and catalytic activity. Both AK1 and AK2 are functional oligomers unlike typical AKs which are monomeric. A phylogenetic analysis showed that these choanoflagellate AKs group more closely with a supercluster consisting of cytoplasmic and mitochondrial CKs and invertebrate AKs that evolved secondarily from a CK-like ancestor. Reaction-diffusion constraints in choanoflagellates are likely mitigated by the presence of AK isoforms which facilitate energy transport in these highly polarized cells.     

Horizontally acquired DAP pathway as a unit of self-regulation

In this study, we report the acquisition of the diaminopimelic acid (DAP) pathway of lysine biosynthesis in choanoflagellate Monosiga brevicollis and investigate how this pathway is incorporated and regulated in the established metabolic network. Our data show that all major genes related to the DAP pathway in Monosiga were acquired from bacteria and algae. Although an endogenous lysC exists in Monosiga, the newly acquired lysC is fused to lysA and used specifically for lysine biosynthesis. In addition, these acquired genes encode two key rate-limiting enzymes, thus conferring Monosiga a self-regulated unit with ability to generate lysine. Our data suggest that a newly acquired metabolic capability can be added to the recipient organism without disturbing the previously established metabolic network. This finding also implies that the biochemical system of the recipient organism may determine the type and function of genes to be acquired.     

PTB domain-directed substrate targeting in a tyrosine kinase from the unicellular choanoflagellate Monosiga brevicollis

Choanoflagellates are considered to be the closest living unicellular relatives of metazoans. The genome of the choanoflagellate Monosiga brevicollis contains a surprisingly high number and diversity of tyrosine kinases, tyrosine phosphatases, and phosphotyrosine-binding domains. Many of the tyrosine kinases possess combinations of domains that have not been observed in any multicellular organism. The role of these protein interaction domains in M. brevicollis kinase signaling is not clear. Here, we have carried out a biochemical characterization of Monosiga HMTK1, a protein containing a putative PTB domain linked to a tyrosine kinase catalytic domain. We cloned, expressed, and purified HMTK1, and we demonstrated that it possesses tyrosine kinase activity. We used immobilized peptide arrays to define a preferred ligand for the third PTB domain of HMTK1. Peptide sequences containing this ligand sequence are phosphorylated efficiently by recombinant HMTK1, suggesting that the PTB domain of HMTK1 has a role in substrate recognition analogous to the SH2 and SH3 domains of mammalian Src family kinases. We suggest that the substrate recruitment function of the noncatalytic domains of tyrosine kinases arose before their roles in autoinhibition.     

动物线粒体基因组变异研究进展

动物mtDNA大多是共价闭合的环状双链分子,一般由2个非编码区和37个编码基因组成,不同动物线粒体基因组大小变异明显.孑遗疟虫(Plasmodium reichenowi)的线粒体基因组最小,仅为5966bp;领鞭毛虫(Monosiga brevicollis)的最大,达76568bp.动物线粒体基因组大小变异的原因主要有:控制区串联重复元件的变异;基因重复;基因重叠与基因间隔区大小的差异;基因缺失和增加.     

Hsp70 sequences indicate that choanoflagellates are closely related to animals.

Over 130 years ago, James-Clark noted a remarkable structural similarity between the feeding cells of sponges (choanocytes) and a group of free-living protists, the choanoflagellates. Both cell types possess a single flagellum surrounded by a collar of fine tentacles. The similarity led to the hypothesis that sponges, and, by implication, other animals, evolved from choanoflagellate-like ancestors. Phylogenetic analysis of ribosomal DNA neither supports nor refutes this hypothesis. Here, we report the sequence of an hsp70 gene and pseudogene from the freshwater choanoflagellate Monosiga ovata. These represent the first nuclear-encoded protein-coding sequences reported for any choanoflagellate. We find that Monosiga and most bilaterian hsp70 genes have high GC contents that may distort phylogenetic tree construction; therefore, protein sequences were used for phylogenetic reconstruction. Our analyses indicate that Monosiga is more closely related to animals than to fungi. We infer that animals and at least some choanoflagellates are part of a clade that excludes the fungi. This is consistent with the origin of animals from a choanoflagellate-like ancestor.     

Mosquitoes LTR retrotransposons: a deeper view into the genomic sequence of Culex quinquefasciatus

A set of 67 novel LTR-retrotransposon has been identified by in silico analyses of the Culex quinquefasciatus genome using the LTR_STRUC program. The phylogenetic analysis shows that 29 novel and putatively functional LTR-retrotransposons detected belong to the Ty3/gypsy group. Our results demonstrate that, by considering only families containing potentially autonomous LTR-retrotransposons, they account for about 1% of the genome of C. quinquefasciatus. In previous studies it has been estimated that 29% of the genome of C. quinquefasciatus is occupied by mobile genetic elements.The potential role of retrotransposon insertions strictly associated with host genes is described and discussed along with the possible origin of a retrotransposon with peculiar Primer Binding Site region. Finally, we report the presence of a group of 38 retrotransposons, carrying tandem repeated sequences but lacking coding potential, and apparently lacking "master copy" elements from which they could have originated. The features of the repetitive sequences found in these non-autonomous LTR retrotransposons are described, and their possible role discussed.These results integrate the existing data on the genomics of an important virus-borne disease vector.     

Molecular evolution of mobile elements of the <Emphasis Type="Italic">gypsy</Emphasis> group: A homolog of the <Emphasis Type="Italic">gag</Emphasis> gene in <Emphasis Type="Italic">Drosophila</Emphasis>

Retrotransposons of the gypsy group of Drosophila melanogaster that are structurally similar to retroviruses of vertebrates occupy an important place among retroelements of eukaryotes. The infectious abilities of some retrotransposons of this group (gypsy, ZAM, and Idefix) have been demonstrated experimentally, and therefore they are true retroviruses. It is supposed that retrotransposons can evolve acquiring new components, the sources of which remain to be elucidated. In this work, the CG4680gene (Gag related protein, Grp) homologous to gag of retrotransposons of the gypsy group has been identified in the genome of D. melanogaster and characterized. The Grp gene product has a highly conserved structure in different species of the Drosophilidae family and is under of purifying selection, which suggests its important genomic function in Drosophila. In view of the earlier data, it can be concluded that homologous genes of all components of gypsy retrotransposons are present in the Drosophila genome. These genes can be both precursors and products of domestication of retrovirus genes.     

The Secretin GPCRs descended from the family of Adhesion GPCRs

The Adhesion G-protein-coupled receptors (GPCRs) are the most complex gene family among GPCRs with large genomic size, multiple introns, and a fascinating flora of functional domains, though the evolutionary origin of this family has been obscure. Here we studied the evolution of all class B (7tm2)-related genes, including the Adhesion, Secretin, and Methuselah families of GPCRs with a focus on nine genomes. We found that the cnidarian genome of Nematostella vectensis has a remarkably rich set of Adhesion GPCRs with a broad repertoire of N-terminal domains although this genome did not have any Secretin GPCRs. Moreover, the single-celled and colony-forming eukaryotes Monosiga brevicollis and Dictyostelium discoideum contain Adhesion-like GPCRs although these genomes do not have any Secretin GPCRs suggesting that the Adhesion types of GPCRs are the most ancient among class B GPCRs. Phylogenetic analysis found Adhesion group V (that contains GPR133 and GPR144) to be the closest relative to the Secretin family in the Adhesion family. Moreover, Adhesion group V sequences in N. vectensis share the same splice site setup as the Secretin GPCRs. Additionally, one of the most conserved motifs in the entire Secretin family is only found in group V of the Adhesion family. We suggest therefore that the Secretin family of GPCRs could have descended from group V Adhesion GPCRs. We found a set of unique Adhesion-like GPCRs in N. vectensis that have long N-termini containing one Somatomedin B domain each, which is a domain configuration similar to that of a set of Adhesion-like GPCRs found in Branchiostoma floridae. These sequences show slight similarities to Methuselah sequences found in insects. The extended class B GPCRs have a very complex evolutionary history with several species-specific expansions, and we identified at least 31 unique N-terminal domains originating from other protein classes. The overall N-terminal domain structure, however, concurs with the phylogenetic analysis of the transmembrane domains, thus enabling us to track the origin of most of the subgroups.     

Vascular smooth muscle myosin light chain diphosphorylation: mechanism, function, and pathological implications

Smooth muscle contraction is activated primarily by phosphorylation at S19 of the 20-kDa regulatory light chain subunits of myosin II (LC(20) ) catalyzed by Ca(2+) /calmodulin-dependent myosin light chain kinase. Other kinases, for example, integrin-linked kinase (ILK), Rho-associated kinase (ROCK), and zipper-interacting protein kinase (ZIPK), can phosphorylate T18 in addition to S19, which increases the actin-activated myosin MgATPase activity at subsaturating actin concentrations ～3-fold. These phosphorylatable residues and the amino acid sequence surrounding them are highly conserved throughout the animal kingdom; they are also found in an LC(20) homolog within the genome of Monosiga brevicollis, the closest living relative of metazoans. LC(20) diphosphorylation has been detected in mammalian vascular smooth muscle tissues in response to specific contractile stimuli and in pathophysiological situations associated with hypercontractility. LC(20) diphosphorylation has also been observed frequently in cultured cells where it activates force generation. Kinases such as ILK, ROCK, and ZIPK, therefore, are potential therapeutic targets in the treatment of, for example, cerebral vasospasm following subarachnoid hemorrhage and atherosclerosis.     

Unicellular Ca2+ signaling 'toolkit' at the origin of metazoa

Ca(2+) signaling pathways control many physiological processes in almost all types of animal cells such as fertilization, muscle contraction, hormone release, and learning and memory. Each animal cell type expresses a unique group of molecules from the Ca(2+) signaling 'toolkit' to control spatiotemporal patterns of Ca(2+) signaling. It is generally believed that the complex Ca(2+) signaling 'toolkit' has arisen from the ancestral multicellular organisms to fit unique physiological roles of specialized cell types. Here, we demonstrate for the first time the presence of an extensive Ca(2+) signaling 'toolkit' in the unicellular choanoflagellate Monosiga brevicollis. Choanoflagellates possess homologues of various types of animal plasma membrane Ca(2+) channels including the store-operated channel, ligand-operated channels, voltage-operated channels, second messenger-operated channels, and 5 out of 6 animal transient receptor potential channel families. Choanoflagellates also contain homologues of inositol 1,4,5-trisphosphate receptors. Furthermore, choanoflagellates master a complete set of Ca(2+) removal systems including plasma membrane and sarco/endoplasmic reticulum Ca(2+) ATPases and homologues of 3 animal cation/Ca(2+) exchanger families. Therefore, a complex Ca(2+) signaling 'toolkit' might have evolved before the emergence of multicellular animals.     

Mitochondrial genomes of two demosponges provide insights into an early stage of animal evolution

Mitochondrial DNA (mtDNA) of multicellular animals (Metazoa) is typically a small ( approximately 16 kbp), circular-mapping molecule that encodes 37 tightly packed genes. The structures of mtDNA-encoded transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs) are usually highly unorthodox, and proteins are translated with multiple deviations from the standard genetic code. In contrast, mtDNA of the choanoflagellate Monosiga brevicollis, the closest unicellular relative of animals, is four times larger, contains 1.5 times as many genes, and lacks mentioned peculiarities of animal mtDNA. To investigate the evolutionary transition that led to the specific organization of metazoan mtDNA, we determined complete mitochondrial sequences from the demosponges Geodia neptuni and Tethya actinia, two representatives of the most basal animal phylum, the Porifera. We found that poriferan mtDNAs resemble those of other animals in their compact organization, lack of introns, and a well-conserved animal-like gene order. Yet, they contain several extra genes, encode bacterial-like rRNAs and tRNAs, and use a minimally derived genetic code. Our findings suggest that the evolution of the typical metazoan mtDNA has been a multistep process in which the compact genome organization and the reduced gene content were established prior to the reduction of tRNA and rRNA structures and the introduction of multiple changes of the translation code.     

The scale and evolutionary significance of horizontal gene transfer in the choanoflagellate Monosiga brevicollis

Background

It is generally agreed that horizontal gene transfer (HGT) is common in phagotrophic protists. However, the overall scale of HGT and the cumulative impact of acquired genes on the evolution of these organisms remain largely unknown.

Results

Choanoflagellates are phagotrophs and the closest living relatives of animals. In this study, we performed phylogenomic analyses to investigate the scale of HGT and the evolutionary importance of horizontally acquired genes in the choanoflagellate Monosiga brevicollis. Our analyses identified 405 genes that are likely derived from algae and prokaryotes, accounting for approximately 4.4% of the Monosiga nuclear genome. Many of the horizontally acquired genes identified in Monosiga were probably acquired from food sources, rather than by endosymbiotic gene transfer (EGT) from obsolete endosymbionts or plastids. Of 193 genes identified in our analyses with functional information, 84 (43.5%) are involved in carbohydrate or amino acid metabolism, and 45 (23.3%) are transporters and/or involved in response to oxidative, osmotic, antibiotic, or heavy metal stresses. Some identified genes may also participate in biosynthesis of important metabolites such as vitamins C and K12, porphyrins and phospholipids.

Conclusions

Our results suggest that HGT is frequent in Monosiga brevicollis and might have contributed substantially to its adaptation and evolution. This finding also highlights the importance of HGT in the genome and organismal evolution of phagotrophic eukaryotes.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-14-729) contains supplementary material, which is available to authorized users.     

Cross-genome screening of novel sequence-specific non-LTR retrotransposons: various multicopy RNA genes and microsatellites are selected as targets

Although most LINEs (long interspersed nuclear elements), which are autonomous non-long-terminal-repeat retrotransposons, are inserted throughout the host genome, three groups of LINEs, the early-branched group, the Tx group, and the R1 clade, are inserted into specific sites within the target sequence. We previously characterized the sequence specificity of the R1 clade elements. In this study, we screened the other two groups of sequence-specific LINEs from public DNA databases, reconstructed elements from fragmented sequences, identified their target sequences, and analyzed them phylogenetically. We characterized 13 elements in the early-branched group and 13 in the Tx group. In the early-branched group, we identified R2 elements from sea squirts and zebrafish in this study, although R2 has not been characterized outside the arthropod group to date. This is the first evidence of cross-phylum distribution of sequence-specific LINEs. The Dong element also occurs across phyla, among arthropods and mollusks. In the Tx group, we characterized five novel sequence-specific families: Kibi for TC repeats, Koshi for TTC repeats, Keno for the U2 snRNA gene, Dewa for the tRNA tandem arrays, and Mutsu for the 5S rRNA gene. Keno and Mutsu insert into the highly conserved region within small RNA genes and destroy the targets. Several copies of Dewa insert different positions of tRNA tandem array, which indicates a certain "site specifier" other than sequence-specific endonuclease. In all three groups, LINEs specific for the rRNA genes or microsatellites can occur as multiple families in one organism. This indicates that the copy number of a target sequence is the primary factor to restrict the variety of sequence specificity of LINEs.     

Phylogenetic analysis of the teneurins: conserved features and premetazoan ancestry

Teneurins are type II transmembrane proteins expressed during pattern formation and neurogenesis with an intracellular domain that can be transported to the nucleus and an extracellular domain that can be shed into the extracellular milieu. In Drosophila melanogaster, Caenorhabditis elegans, and mouse the knockdown or knockout of teneurin expression can lead to abnormal patterning, defasciculation, and abnormal pathfinding of neurites, and the disruption of basement membranes. Here, we have identified and analyzed teneurins from a broad range of metazoan genomes for nuclear localization sequences, protein interaction domains, and furin cleavage sites and have cloned and sequenced the intracellular domains of human and avian teneurins to analyze alternative splicing. The basic organization of teneurins is highly conserved in Bilateria: all teneurins have epidermal growth factor (EGF) repeats, a cysteine-rich domain, and a large region identical in organization to the carboxy-half of prokaryotic YD-repeat proteins. Teneurins were not found in the genomes of sponges, cnidarians, or placozoa, but the choanoflagellate Monosiga brevicollis has a gene encoding a predicted teneurin with a transmembrane domain, EGF repeats, a cysteine-rich domain, and a region homologous to YD-repeat proteins. Further examination revealed that most of the extracellular domain of the M. brevicollis teneurin is encoded on a single huge 6,829-bp exon and that the cysteine-rich domain is similar to sequences found in an enzyme expressed by the diatom Phaeodactylum tricornutum. This leads us to suggest that teneurins are complex hybrid fusion proteins that evolved in a choanoflagellate via horizontal gene transfer from both a prokaryotic gene and a diatom or algal gene, perhaps to improve the capacity of the choanoflagellate to bind to its prokaryotic prey. As choanoflagellates are considered to be the closest living relatives of animals, the expression of a primitive teneurin by an ancestral choanoflagellate may have facilitated the evolution of multicellularity and complex histogenesis in metazoa.