Retrotransposons and Tandem Repeat Sequences in the Nuclear Genomes of Cryptomonad Algae

The cryptomonads are an enigmatic group of unicellular eukaryotic algae that possess two nuclear genomes, having acquired photosynthesis by the uptake and retention of a eukaryotic algal endosymbiont. The endosymbiont nuclear genome, or nucleomorph, of the cryptomonad Guillardia theta has been completely sequenced: at only 551 kilobases (kb) and with a gene density of ∼1 gene/kb, it is a model of compaction. In contrast, very little is known about the structure and composition of the cryptomonad host nuclear genome. Here we present the results of two small-scale sequencing surveys of fosmid clone libraries from two distantly related cryptomonads, Rhodomonas salina CCMP1319 and Cryptomonas paramecium CCAP977/2A, corresponding to ∼150 and ∼235 kb of sequence, respectively. Very few of the random end sequences determined in this study show similarity to known genes in other eukaryotes, underscoring the considerable evolutionary distance between the cryptomonads and other eukaryotes whose nuclear genomes have been completely sequenced. Using a combination of fosmid clone end-sequencing, Southern hybridizations, and PCR, we demonstrate that Ty3-gypsy long-terminal repeat (LTR) retrotransposons and tandem repeat sequences are a prominent feature of the nuclear genomes of both organisms. The complete sequence of a 30.9-kb genomic fragment from R. salina was found to contain a full-length Ty3-gypsy element with near-identical LTRs and a chromodomain, a protein module suggested to mediate the site-specific integration of the retrotransposon. The discovery of chromodomain-containing retroelements in cryptomonads further expands the known distribution of the so-called chromoviruses across the tree of eukaryotes. [Reviewing Editor: Dr. Debashish Bhattacharya]     

Impacts of gene essentiality, expression pattern, and gene compactness on the evolutionary rate of mammalian proteins

Understanding the determinants of the rate of protein sequenceevolution is of fundamental importance in evolutionary biology.Many recent studies have focused on the yeast because of theavailability of many genome-wide expressional and functionaldata. Yeast studies revealed a predominant role of gene expressionlevel and a minor role of gene essentiality in determining therate of protein sequence evolution. Whether these rules applyto complex organisms such as mammals is unclear. Here we assemblea list of 1,138 essential and 2,341 nonessential mouse genesbased on targeted gene deletion experiments and report a significantimpact of gene essentiality on the rate of mammalian proteinevolution. Gene expression level has virtually no effect, althoughtissue specificity in expression pattern has a strong influence.Unexpectedly, gene compactness, measured by average intron sizeand untranslated region length, has the greatest influence.Hence, the relative importance of the various factors in determiningthe rate of mammalian protein evolution is gene compactness> gene essentiality tissue specificity > expression level.Our results suggest a considerable variation in rate determinantsbetween unicellular organisms such as the yeast and multicellularorganisms such as mammals.     

Low rates of expression profile divergence in highly expressed genes and tissue-specific genes during mammalian evolution

Evolutionary rates provide important information about the pattern and mechanism of evolution. Although the rate of gene sequence evolution has been well studied, the rate of gene expression evolution is poorly understood. In particular, it is unclear whether the gene expression level and tissue specificity influence the divergence of expression profiles between orthologous genes. Here we address this question using a microarray data set comprising the expression signals of 10,607 pairs of orthologous human and mouse genes from over 60 tissues per species. We show that the level of gene expression and the degree of tissue specificity are generally conserved between the human and mouse orthologs. The rate of gene expression profile change during evolution is negatively correlated with the level of gene expression, measured by either the average or the highest level among all tissues examined. This is analogous to the observation that the rate of gene (or protein) sequence evolution is negatively correlated with the gene expression level. The impacts of the degree of tissue specificity on the evolutionary rate of gene sequence and that of expression profile, however, are opposite. Highly tissue-specific genes tend to evolve rapidly at the gene sequence level but slowly at the expression profile level. Thus, different forces and selective constraints must underlie the evolution of gene sequence and that of gene expression.     

The mammalian PYHIN gene family: Phylogeny, evolution and expression

ABSTRACT: BACKGROUND: Proteins of the mammalian PYHIN (IFI200/HIN-200) family are involved in defence against infection through recognition of foreign DNA. The family member absent in melanoma 2 (AIM2) binds cytosolic DNA via its HIN domain and initiates inflammasome formation via its pyrin domain. AIM2 lies within a cluster of related genes, many of which are uncharacterised in mouse. To better understand the evolution, orthology and function of these genes, we have documented the range of PYHIN genes present in representative mammalian species, and undertaken phylogenetic and expression analyses. RESULTS: No PYHIN genes are evident in non-mammals or monotremes, with a single member found in each of three marsupial genomes. Placental mammals show variable family expansions, from one gene in cow to four in human and 14 in mouse. A single HIN domain appears to have evolved in the common ancestor of marsupials and placental mammals, and duplicated to give rise to three distinct forms (HIN-A, -B and -C) in the placental mammal ancestor. Phylogenetic analyses showed that AIM2 HIN-C and pyrin domains clearly diverge from the rest of the family, and it is the only PYHIN protein with orthology across many species. Interestingly, although AIM2 is important in defence against some bacteria and viruses in mice, AIM2 is a pseudogene in cow, sheep, llama, dolphin, dog and elephant. The other 13 mouse genes have arisen by duplication and rearrangement within the lineage, which has allowed some diversification in expression patterns. CONCLUSIONS: The role of AIM2 in forming the inflammasome is relatively well understood, but molecular interactions of other PYHIN proteins involved in defence against foreign DNA remain to be defined. The non-AIM2 PYHIN protein sequences are very distinct from AIM2, suggesting they vary in effector mechanism in response to foreign DNA, and may bind different DNA structures. The PYHIN family has highly varied gene composition between mammalian species due to lineage-specific duplication and loss, which probably indicates different adaptations for fighting infectious disease. Non-genomic DNA can indicate infection, or a mutagenic threat. We hypothesise that defence of the genome against endogenous retroelements has been an additional evolutionary driver for PYHIN proteins.     

Ribonucleotide reductases: the evolution of allosteric regulation.

Ribonucleotide reductases catalyze in all living organisms the production of the deoxyribonucleotides required for DNA replication and repair. Their appearance during evolution was a prerequisite for the transition from the "RNA world," where RNA sufficed for both catalysis and information transfer, to today's situation where life depends on the interplay among DNA, RNA, and protein. Three classes of ribonucleotide reductases exist today, widely differing in their primary and quaternary structures but all with a highly similar allosteric regulation of their substrate specificity. Here, I discuss the diversities between the three classes, describe their allosteric regulation, and discuss the evidence for their evolution. The appearance of oxygen on earth provided the likely driving force for enzyme diversification. From today's characteristics of the three classes, including their allosteric regulation, I propose that the anaerobic class III reductases with their iron-sulfur cluster and the requirement for S-adenosylmethionine for the generation of a glycyl protein free radical are the closest relatives to an ancestor ribonucleotide reductase.     

Three novel mammalian toll-like receptors: gene structure, expression, and evolution

We describe three novel genes, encoding members of the Toll-like receptor (Tlr) family (TLR7, TLR8, and TLR9). These Tlr family members, unlike others reported to date, were identified within a genomic database. TLR7 and TLR8 each have three exons, two of which have coding function, and lie in close proximity to one another at Xp22, alongside a pseudogene. The remaining gene (TLR9) resides at 3p21.3 (in linkage with the MyD88 gene), and is expressed in at least two splice forms, one of which is monoexonic and one of which is biexonic, the latter encoding a protein with 57 additional amino acids at the N-terminus. The novel Tlrs comprise a cluster as nearest phylogenetic neighbors. Combining all sequence data related to Toll-like receptors, we have drawn several inferences concerning the phylogeny of vertebrate and invertebrate Tlrs. According to our best estimates, mammalian TLRs 1 and 6 diverged from a common mammalian ancestral gene 95 million years ago. TLR4, which encodes the endotoxin sensor in present-day mammals, emerged as a distinct entity 180 million years ago. TLRs 3 and 5 diverged from a common ancestral gene approximately 150 million years ago, as did Tlr7 and Tlr8. Very likely, fewer Tlrs existed during early vertebrate evolution: at most three or four were transmitted with the primordial vertebrate line. Phylogenetic data that we have adduced in the course of this work also suggest the existence of a Drosophila equivalent of MyD88, and indicate that the plasma membrane protein SIGIRR is close functional relative of MyD88 in mammals. Finally, a single present-day representative of the Toll-like proteins in Drosophila has striking cytoplasmic domain homology to mammalian Tlrs within the cluster that embraces TLRs 1, 2, 4, and 6. This would suggest that an ancestral (pre-vertebrate) Tlr may have adopted a pro-inflammatory function 500 million years ago.     

Characterization of the Ferredoxin-Gogat gene (OsGog2 clone) expression in rice

Ferredoxin-dependent glutamate synthase (Fd-Gogat; EC 1.4.7.1) in leaf and root plastids is the last enzyme involved in the pathway of nitrate assimilation in higher plants. Arabidopsis thaliana expresses two different genes: the first, light regulated, specific of green tissues and the second expressed in other tissues. In this work, we investigated whether in our clone, OsGog2 AC Y12595, this gene is up-regulated by light or it is expressed under darkness. Fd-Gogat specific activity, protein and mRNA increased after light treatment in rice shoots. In roots, the activity and the protein content remained constant, whereas the mRNA is repressed by light treatment. The results obtained using a specific probe, situated in the 3′ untranslated region of the OsGog2 cDNA, indicated that OsGog2 gene is up-regulated by light and that its expression is tissue specific and suggested that a dark expressed Fd-Gogat gene could be present in rice similarly as in Arabidopsis.     

Expression and evolution of the mammalian brain gene Ttyh1

Homologues of the Drosophila melanogaster tweety (tty) gene are present in mammals and Caenorhabditis elegans. The encoded proteins have five predicted membrane-spanning regions and recent findings suggest that some family members may be chloride channels. Phylogenetic analysis of the tty family including novel members from slime mould Entamoeba and plants has revealed the occurrence of independent gene duplication events in different lineages. expressed sequence tag data indicate that expression of the mammalian Ttyh1 gene is restricted mainly to neural tissue and is up-regulated in astrocytoma, glioma and several other cancers. In this study, mammalian expression vectors were used to investigate the subcellular localization and the effect of over-expression of Ttyh1 in human epithelial kidney cells. The results confirm that Ttyh1 is a membrane protein and show that it is deposited on the substratum along the migration paths of motile cells above the alpha5beta1-integrin complex. The ectopic expression of Ttyh1 also induced long filopodia, which were branched and dynamic in both stationary and migratory cells. The filopodia contained F-actin and occurred at the ends of microtubules which were polarized towards the membrane. Upon contact with nearby cells some filopodia stabilized and filled with F-actin, whereas Ttyh1 was highly concentrated at the cell-cell interface. Ttyh1 N- and C-terminal antipeptide antibodies detected Ttyh1 along the axons of neurones in primary rat hippocampal cell cultures, and in situ in whole rat brain slices around the hippocampus and occasionally between cells. These data suggest a role for Ttyh1 in process formation, cell adhesion and possibly as a transmembrane receptor.     

Developmental and regional-specific expression of FLRFamide peptides in the tobacco hornworm,Manduca sexta,suggests functions at ecdysis

The developmental profile of a family of three FLRFamide (Phe-Leu-Arg-Phe-NH₂) peptides in the tobacco hornworm, Manduca sexta, revealed regional-specific expression patterns within the segmental ganglia. Levels of the three peptides—F7G (GNSFLRFamide), F7D (DPSFLRFamide), and F10 (pEDVVHSFLRFamide)—were always higher in the thoracic than abdominal ganglia. The predominant peptide also differed regionally, with F7G being highest in the thoracic ganglia and F7G and F10 being equivalent in the abdominal ganglia. Furthermore, we found regional-specific transient declines in ganglion peptide levels temporally correlated to ecdysis. Thoracic ganglion peptide levels declined at each molt, while abdominal ganglion levels declined over a period of 2 days after ecdysis. The decline in central levels was accompanied by an increase in levels in peripheral neurohemal sites, the transverse nerves (TNs). These observations suggest peptides were released from neurosecretory cells (NSCs) at ecdysis. Distinct sets of thoracic and abdominal NSCs and their processes in peripheral neurohemal sites were immunoreactive, supporting the biochemical data. These results also suggest the regional differences may arise from cellular-specific expression patterns for this family of peptides. In addition, fine immunoreactive processes were observed traveling between TNs and skeletal muscles, suggestive of myotropic actions. We propose that the release of different M. sexta FLRFamides from regionally distinct NSCs leads to a coordinated modulation of skeletal and visceral muscles that facilitate ecdysis. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 469–485, 1998     

Predominant expression of a peroxidase gene in staminate flowers of Mercurialis annua

A direct correlation between the feminizing process of male plants of Mercurialis annua by benzylaminopurine and the disappearance of male-specific peroxidases was established. In order to understand the process of hormonal regulation of the sexual differentiation and peroxidase activity, two peroxidase cDNA clones were selected from a male flower cDNA library with degenerated oligonucleotide probes encoding a peroxidase-specific site. These clones contained three peroxidase-specific sequences and an Eco RI restriction site. The two Eco RI fragments of one cDNA clone were subcloned and used as probes to analyse the expression of corresponding gene(s). The northern blot analysis showed hybridization on a single mRNA band with a predominant expression in male flowers. The feminizing treatment with a cytokinin induced the progressive disappearance of the hybridization signal in correlation with the disappearance of male specific isoperoxidases.     

Germ cell-specific expression of a proacrosin-CAT fusion gene in transgenic mouse testis.

Acrosin is a serine proteinase located in a zymogen form, proacrosin in the acrosome of the sperm. It is released as a consequence of the acrosome reaction and is believed to be the most important enzyme in the fertilization process. In the mouse, the proacrosin gene is transcribed premeiotically in spermatocytes, but protein biosynthesis starts in haploid spermatids and is restricted to the emerging acrosome. Four lines of transgenic mice harboring 2.3 kb of 5' untranslated region of the rat proacrosin gene fused to the CAT-reporter gene were generated by microinjection of fertilized eggs. The chimeric gene was found to be present in 10-100 copies per genome in the different strains. The 5' untranslated region of rat proacrosin gene could properly direct CAT-gene expression to spermatocytes and CAT-mRNA translation to round spermatids as it is known for mouse proacrosin gene. However, CAT protein is not restricted to the acrosome; rather, it is distributed in the spermatid cytoplasm. This could be due to the lack of DNA sequences for a hydrophobic leader peptide that have been found in all mammalian proacrosins studied until now but that was not present in transgene. It can be concluded from our results that cis-acting sequences required for tissue specific proacrosin expression reside on a 2.3-kb restriction fragment and are conserved in the proacrosin genes of mouse and rat.