The ingi and RIME non-LTR retrotransposons are not randomly distributed in the genome of Trypanosoma brucei

The ingi (long and autonomous) and RIME (short and nonautonomous) non--long-terminal repeat retrotransposons are the most abundant mobile elements characterized to date in the genome of the African trypanosome Trypanosoma brucei. These retrotransposons were thought to be randomly distributed, but a detailed and comprehensive analysis of their genomic distribution had not been performed until now. To address this question, we analyzed the ingi/RIME sequences and flanking sequences from the ongoing T. brucei genome sequencing project (TREU927/4 strain). Among the 81 ingi/RIME elements analyzed, 60% are complete, and 7% of the ingi elements (approximately 15 copies per haploid genome) appear to encode for their own transposition. The size of the direct repeat flanking the ingi/RIME retrotransposons is conserved (i.e., 12-bp), and a strong 11-bp consensus pattern precedes the 5'-direct repeat. The presence of a consensus pattern upstream of the retroelements was confirmed by the analysis of the base occurrence in 294 GSS containing 5'-adjacent ingi/RIME sequences. The conserved sequence is present upstream of ingis and RIMEs, suggesting that ingi-encoded enzymatic activities are used for retrotransposition of RIMEs, which are short nonautonomous retroelements. In conclusion, the ingi and RIME retroelements are not randomly distributed in the genome of T. brucei and are preceded by a conserved sequence, which may be the recognition site of the ingi-encoded endonuclease.     

Long-term inheritance of the 28S rDNA-specific retrotransposon R2

R2 is a non-long-terminal-repeat (LTR) retrotransposon that inserts specifically into 28S rDNA. R2 has been identified in many species of arthropods and three species of chordates. R2 may be even more widely distributed in animals, and its origin may be traceable to early animal evolution. In this study, we identified R2 elements in medaka fish, White Cloud Mountain minnow, Reeves' turtle, hagfish, sea lilies, and some arthropod species, using degenerate polymerase chain reaction methods. We also identified two R2 elements from the public genomic sequence database of the bloodfluke Schistosoma mansoni. One of the two bloodfluke R2 elements has two zinc-finger motifs at the N-terminus; this differs from other known R2 elements, which have one or three zinc-finger motifs. Phylogenetic analysis revealed that the whole phylogeny of R2 can be divided into 11 parts (subclades), in which the local R2 phylogeny and the corresponding host phylogeny are consistent. Divergence-versus-age analysis revealed that there is no reliable evidence for the horizontal transfer of R2 but supports the proposition that R2 has been vertically transferred since before the divergence of the deuterostomes and protostomes. The seeming inconsistency between the R2 phylogeny and the phylogeny of their hosts is due to the existence of paralogous lineages. The number of N-terminal zinc-finger motifs is consistent with the deep phylogeny of R2 and indicates that the common ancestor of R2 had three zinc-finger motifs at the N-terminus. This study revealed the long-term vertical inheritance and the ancient origin of sequence specificity of R2, both of which seem applicable to some other non-LTR retrotransposons.     

RetroPred: A tool for prediction, classification and extraction of non-LTR retrotransposons (LINEs & SINEs) from the genome by integrating PALS, PILER, MEME and ANN

The problem of predicting non-long terminal repeats (LTR) like long interspersed nuclear elements (LINEs) and short interspersed nuclear elements (SINEs) from the DNA sequence is still an open problem in bioinformatics. To elevate the quality of annotations of LINES and SINEs an automated tool "RetroPred" was developed. The pipeline allowed rapid and thorough annotation of non-LTR retrotransposons. The non-LTR retrotransposable elements were initially predicted by Pairwise Aligner for Long Sequences (PALS) and Parsimonious Inference of a Library of Elementary Repeats (PILER). Predicted non-LTR elements were automatically classified into LINEs and SINEs using ANN based on the position specific probability matrix (PSPM) generated by Multiple EM for Motif Elicitation (MEME). The ANN model revealed a superior model (accuracy = 78.79 +/- 6.86 %, Q(pred) = 74.734 +/- 17.08 %, sensitivity = 84.48 +/- 6.73 %, specificity = 77.13 +/- 13.39 %) using four-fold cross validation. As proof of principle, we have thoroughly annotated the location of LINEs and SINEs in rice and Arabidopsis genome using the tool and is proved to be very useful with good accuracy. Our tool is accessible at http://www.juit.ac.in/RepeatPred/home.html.     

A high-density cytogenetic map of the Aegilops tauschii genome incorporating retrotransposons and defense-related genes: insights into cereal chromosome structure and function

Aegilops tauschii (Coss.) Schmal. (2n=2x=14, DD) (syn. A. squarrosa L.; Triticum tauschii) is well known as the D-genome donor of bread wheat (T. aestivum, 2n=6x=42, AABBDD). Because of conserved synteny, a high-density map of the A. tauschii genome will be useful for breeding and genetics within the tribe Triticeae which besides bread wheat also includes barley and rye. We have placed 249 new loci onto a high-density integrated cytological and genetic map of A. tauschii for a total of 732 loci making it one of the most extensive maps produced to date for the Triticeae species. Of the mapped loci, 160 are defense-related genes. The retrotransposon marker system recently developed for cultivated barley (Hordeum vulgare L.) was successfully applied to A. tauschii with the placement of 80 retrotransposon loci onto the map. A total of 50 microsatellite and ISSR loci were also added. Most of the retrotransposon loci, resistance (R), and defense-response (DR) genes are organized into clusters: retrotransposon clusters in the pericentromeric regions, R and DR gene clusters in distal/telomeric regions. Markers are non-randomly distributed with low density in the pericentromeric regions and marker clusters in the distal regions. A significant correlation between the physical density of markers (number of markers mapped to the chromosome segment/physical length of the same segment in m) and recombination rate (genetic length of a chromosome segment/physical length of the same segment in m) was demonstrated. Discrete regions of negative or positive interference (an excess or deficiency of crossovers in adjacent intervals relative to the expected rates on the assumption of no interference) was observed in most of the chromosomes. Surprisingly, pericentromeric regions showed negative interference. Islands with negative, positive and/or no interference were present in interstitial and distal regions. Most of the positive interference was restricted to the long arms. The model of chromosome structure and function in cereals with large genomes that emerges from these studies is discussed.     

Of babies and birds: complex tool behaviours are not sufficient for the evolution of the ability to create a novel causal intervention

Humans are capable of simply observing a correlation between cause and effect, and then producing a novel behavioural pattern in order to recreate the same outcome. However, it is unclear how the ability to create such causal interventions evolved. Here, we show that while 24-month-old children can produce an effective, novel action after observing a correlation, tool-making New Caledonian crows cannot. These results suggest that complex tool behaviours are not sufficient for the evolution of this ability, and that causal interventions can be cognitively and evolutionarily disassociated from other types of causal understanding.     

Restriction endonuclease mapping of ribosomal RNA genes: Sequence divergence and the origin of the tetraploid treefrog Hyla versicolor

Hyla chrysoscelis (2n=24) and H. versicolor (2n=48) are a diploid-tetraploid species pair of treefrogs. Restriction endonuclease mapping of ribosomal RNA (rRNA) gene repeat units of diploids collected from eastern and western populations reveals no differences within rRNA gene coding regions but distinctive differences within the nontranscribed spacers. A minimum of two physical maps is required to construct an rRNA gene map for the tetraploid, whose repeat units appear to be a composite, with about 50% of the elements resembling the western diploid population and about 50% resembling the eastern population. These results imply that this population of the tetraploid species may have arisen from a genetically hybrid diploid. Alternatively, the dual level of sequence heterogeneity in H. versicolor may reflect some type of gene flow between the two species. The coding region of the rRNA genes in the tetraploid differs from that in either diploid in about 20% of all repeat units, as exemplified by a BamHI site located near the 5 terminus of the 28 S rRNA gene. If the 20% variant class of 28 S rRNA gene coding sequences is expressed, then there must be two structural classes of ribosomes; if only the 80% sequence class is expressed, then a genetic control mechanism must be capable of distinguishing between the two different sequence variants. It is postulated that the 20% variant sequence class may be correlated with a partial functional diploidization of rRNA genes in the tetraploid species.This research was supported, in part, by NSF Grants CDP-8002341 and PRM-8106947 and by faculty research grants from Miami University to J.C.V.     

Characterization of the U3 and U6 snRNA genes from wheat: U3 snRNA genes in monocot plants are transcribed by RNa polymerase III

We have demonstrated recently that the genes encoding the U3 small nuclear RNA (snRNA) in dicot plants are transcribed by RNA polymerase III (pol III), and not RNA polymerase II (pol II) as in all other organisms studied to date. The U3 gene was the first example of a gene transcribed by different polymerases in different organisms. Based on phylogenetic arguments we proposed that a polymerase specificity change of the U3 snRNA gene promoter occurred during plant evolution. To map such an event we are examining the U3 gene polymerase specificity in other plant species. We report here the characterization of a U3 gene from wheat, a monocot plant. This gene contains the conserved promoter elements, USE and TATA, in a pol III-specific spacing seen also in a wheat U6 snRNA gene characterized in this report. Both the U3 and the U6 genes possess typical pol III termination signals but lack the cis element, responsible for 3-end formation, found in all plant pol II-specific snRNA genes. In addition, expression of the U3 gene in transfected maize protoplasts is less sensitive to -amanitin than a pol II-transcribed U2 gene. Based on these data we conclude that the wheat U3 gene is transcribed by pol III. This observation suggests that the postulated RNA polymerase specificity switch of the U3 gene took place prior to the divergence of angiosperm plants into monocots and dicots.     

Enzyme-coding genes as molecular clocks: The molecular evolution of animal alpha-amylases

Summary We constructed a cDNA library for the beetle,Tribolium castaneum. This library was screened using a cloned amylase gene fromDrosophila melanogaster as a molecular probe. Beetle amylase cDNA clones were isolated from this bank, and the nucleotide sequence was obtained for a cDNA clone with a coding capacity for 228 amino acids. Both the nucleotide sequence and predicted amino acid sequence were compared to our recent results forD. melanogaster alpha-amylases, along with published sequences for other alpha-amylases. The results show that animal alpha-amylases are highly conserved over their entire length. A borader comparison, which includes plant and microbial alpha-amylase sequences, indicates that parts of the gene are conserved between prokaryotes, plants, and animals. We discuss the potential importance of this and other enzyme-coding genes for the construction of molecular phylogenies and for the study of the general question of molecular clocks in evolution.     

Identification of novel tropomyosin 1 genes of pufferfish (Fugu rubripes) on genomic sequences and tissue distribution of their transcripts

Fugu genome database enabled us to identify two novel tropomyosin 1 (TPM1) genes through in silico data mining and isolation of their corresponding cDNAs in vivo. The duplicate TPM1 genes in Japanese pufferfish Fugu rubripes suggest that additional an ancient segmental duplication or whole genome duplication occurred in fish lineage, which, like many other reported Fugu genes, showed reduction in genomic size in comparison with their human homologue. Computer analysis predicted that the coiled-coil probabilities, that were thought to be the most major function of TPM, were the same between the two TPM1 isoforms. We confirmed that the tissue expression profiles of the two TPM1 genes differed from each other, which implied that changes in expression pattern could fix duplicated TPM1 genes although the two TPM1 isoforms appear to have similar function.     

Molecular cloning and characterization of ribosomal RNA genes from the brine shrimp: Nucleotide sequence analysis and evolution of the 5.8 S rRNA gene region and its flanking nucleotides

A detailed restriction endonuclease map was prepared for the cloned 5.8 S ribosomal RNA (rRNA) gene region of the brine shrimp Artemia. The nucleotide sequence of the 5.8 S rRNA gene and its flanking nucleotides was determined. This sequence differs in two positions from that of the previously reported 5.8 S rRNA. The primary structure of the Artemia 5.8 S rRNA gene, which, unlike in dipteran insects, is shown to contain no insertion sequence, is conserved according to the relatedness of the species compared. The 5.8 S rRNA gene flanking nucleotides, which were sequenced 176 nucleotide pairs upstream and 70 nucleotide pairs downstream from the gene, show no evidence of sequence conservation between evolutionarily diverse species by computer analysis. Direct nucleotide repeats are present within the flanking sequences at both ends of the gene at about the same distance upstream and downstream, which could serve as processing signals.     

Single nucleotide polymorphisms in genes that are common targets of luteotropin and luteolysin in primate corpus luteum: Computational exploration

Luteal insufficiency affects fertility and hence study of mechanisms that regulate corpus luteum (CL) function is of prime importance to overcome infertility problems. Exploration of human genome sequence has helped to study the frequency of single nucleotide polymorphisms (SNPs). Clinical benefits of screening SNPs in infertility are being recognized well in recent times. Examining SNPs in genes associated with maintenance and regression of CL may help to understand unexplained luteal insufficiency and related infertility. Publicly available microarray gene expression databases reveal the global gene expression patterns in primate CL during the different functional state. We intend to explore computationally the deleterious SNPs of human genes reported to be common targets of luteolysin and luteotropin in primate CL. Different computational algorithms were used to dissect out the functional significance of SNPs in the luteinizing hormone sensitive genes. The results raise the possibility that screening for SNPs might be integrated to evaluate luteal insufficiency associated with human female infertility for future studies.     

A BC200-derived element and Z-DNA as structural markers in annexin I genes: Relevance to Alu evolution and annexin tetrad formation

We have identified two types of structural elements in genomic DNA for annexin I that provide physical evidence of genetic events leading to conserved changes in gene structure. The sequence upstream of the transcribed region in human annexin I contained a rare, Alu-like repetitive element with flanking direct repeats, probably derived from the active BC200 gene via germline retroposition. Nucleotide substitutions in this BC200 insert relative to the 7SL gene and its absence in rodent annexins I identified it as a recent primate pseudogene. Phylogenetic analysis showed that the BC200 gene represents a new clade of primate Alu evolution that branched near the time of appearance of the progenitor to the free left Alu monomer, FLAM-C. Separate analysis identified a Z-DNA motif in pigeon annexin I intron 7 that may represent the vestigial recombination site involved in primordial assembly of the annexin tetrad. These distinct structural features in annexin I genes provide insight into the evolution of Alu repeats and the mechanism of annexin tetrad formation.     

Two classes of differentially regulated glutamine synthetase genes are expressed in the soybean nodule: a nodule-specific class and a constitutively expressed class

We have characterized two sets of cDNA clones representing the glutamine synthetase (GS) mRNA in soybean nodules. Using the 3-untranslated regions of a representative member of each set, as gene member(s) specific probes, we have shown that one set of the GS genes are expressed in a nodule-specific manner, while the other set is expressed in other tissues, besides the nodules. The nodule-specific GS genes are expressed in a developmentally regulated manner in the nodules, independent of the onset of nitrogen fixation. The other class of GS genes is expressed constitutively in all tissues tested, but its expression level is dramatically enhanced in nodules following onset of N2 fixation. The latter set of genes is also expressed in cotyledons of germinating seedlings in a developmentally regulated manner. Analysis of hybrid select translation products and genomic Southern blots suggests that multiple gene members in each class are expressed in the nodules.     

Editor's choice: AnABlast: a new in silico strategy for the genome-wide search of novel genes and fossil regions

Genome annotation, assisted by computer programs, is one of the great advances in modern biology. Nevertheless, the in silico identification of small and complex coding sequences is still challenging. We observed that amino acid sequences inferred from coding—but rarely from non-coding—DNA sequences accumulated alignments in low-stringency BLAST searches, suggesting that this alignments accumulation could be used to highlight coding regions in sequenced DNA. To investigate this possibility, we developed a computer program (AnABlast) that generates profiles of accumulated alignments in query amino acid sequences using a low-stringency BLAST strategy. To validate this approach, all six-frame translations of DNA sequences between every two annotated exons of the fission yeast genome were analysed with AnABlast. AnABlast-generated profiles identified three new copies of known genes, and four new genes supported by experimental evidence. New pseudogenes, ancestral carboxyl- and amino-terminal subtractions, complex gene rearrangements, and ancient fragments of mitDNA and of bacterial origin, were also inferred. Thus, this novel in silico approach provides a powerful tool to uncover new genes, as well as fossil-coding sequences, thus providing insight into the evolutionary history of annotated genomes.     

Discovery of novel purine nucleoside derivatives as phosphodiesterase 2 (PDE2) inhibitors: Structure-based virtual screening,optimization and biological evaluation

Phosphodiesterase 2 (PDE2) has received much attention for the potential treatment of the central nervous system (CNS) disorders and pulmonary hypertension. Herein, we identified that clofarabine (4), an FDA-approved drug, displayed potential PDE2 inhibitory activity (IC₅₀?=?3.12?±?0.67?μM) by structure-based virtual screening and bioassay. Considering the potential therapeutic benefit of PDE2, a series of purine nucleoside derivatives based on the structure and binding mode of 4 were designed, synthesized and evaluated, which led to the discovery of the best compound 14e with a significant improvement of inhibitory potency (IC₅₀?=?0.32?±?0.04?μM). Further molecular docking and molecular dynamic (MD) simulations studies revealed that 5′-benzyl group of 14e could interact with the unique hydrophobic pocket of PDE2 by forming extra van der Waals interactions with hydrophobic residues such as Leu770, Thr768, Thr805 and Leu809, which might contribute to its enhancement of PDE2 inhibition. These potential compounds reported in this article and the valuable structure-activity relationships (SARs) might bring significant instruction for further development of potent PDE2 inhibitors.     

Evolution of a pseudogene: exclusive survival of a functional mitochondrial nad7 gene supports Haplomitrium as the earliest liverwort lineage and proposes a secondary loss of RNA editing in Marchantiidae

Gene transfer from the mitochondrion into the nucleus is a corollary of the endosymbiont hypothesis. The frequent and independent transfer of genes for mitochondrial ribosomal proteins is well documented with many examples in angiosperms, whereas transfer of genes for components of the respiratory chain is a rarity. A notable exception is the nad7 gene, encoding subunit 7 of complex I, in the liverwort Marchantia polymorpha, which resides as a full-length, intron-carrying and transcribed, but nonspliced pseudogene in the chondriome, whereas its functional counterpart is nuclear encoded. To elucidate the patterns of pseudogene degeneration, we have investigated the mitochondrial nad7 locus in 12 other liverworts of broad phylogenetic distribution. We find that the mitochondrial nad7 gene is nonfunctional in 11 of them. However, the modes of pseudogene degeneration vary: whereas point mutations, accompanied by single-nucleotide indels, predominantly introduce stop codons into the reading frame in marchantiid liverworts, larger indels introduce frameshifts in the simple thalloid and leafy jungermanniid taxa. Most notably, however, the mitochondrial nad7 reading frame appears to be intact in the isolated liverwort genus Haplomitrium. Its functional expression is shown by cDNA analysis identifying typical RNA-editing events to reconstitute conserved codon identities and also confirming functional splicing of the 2 liverwort-specific group II introns. We interpret our results 1) to indicate the presence of a functional mitochondrial nad7 gene in the earliest land plants and strongly supporting a basal placement of Haplomitrium among the liverworts, 2) to indicate different modes of pseudogene degeneration and chondriome evolution in the later branching liverwort clades, 3) to suggest a surprisingly long maintenance of a nonfunctional gene in the presumed oldest group of land plants, and 4) to support the model of a secondary loss of RNA-editing activity in marchantiid liverworts.