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The retrotransposons HeT-A, TART, and TAHRE, which maintain Drosophila telomeres, transpose specifically onto chromosome ends to form long arrays that extend the chromosome and compensate for terminal loss. Because they transpose by target-primed reverse transcription, each element is oriented so that its 5' end serves as the extreme end of the chromosome until another element transposes to occupy the terminal position. Thus 5' sequences are at risk for terminal erosion while the element is at the chromosome end. Here we report that TART elements in Drosophila melanogaster and Drosophila virilis show species-specific innovations in promoter architecture that buffer loss of sequence exposed at chromosome ends. The two elements have evolved different ways to effect this protection. The D. virilis TART ([TART.sup.vir]) promoter is found in the 3' UTR of the element directly upstream of the element transcribed. Transcription starts within the upstream element so that a 'Tag' of extra sequence is added to the 5' end of the newly transcribed RNA. This Tag provides expendable sequence to buffer end erosion of essential 5' sequence after the RNA is reverse transcribed onto the chromosome. In contrast, the D. melanogaster TART ([TART.sup.mel]) promoter initiates transcription deep within the 5' UTR, but the element is able to replace and extend the 5' UTR sequence by copying sequence from its 3' UTR, we believe while being reverse transcribed onto the chromosome end. Astonishingly, end-protection in [TART.sup.vir] and [HeT-A.sup.mel] are essentially identical (using Tags), whereas [HeT-A.sup.vir] is clearly protected from end erosion by an as-yet-unspecified program. chromosome end protection | promoter evolution | pseudo-LTR promoter | perfect nonterminal repeat | antisense RNA doi/ 10.1073/pnas.1015926107

The non-LTR retrotransposons forming Drosophila telomeres constitute a robust mechanism for telomere maintenance, one which has persisted since before separation of the extant Drosophila species. These elements in D. melanogaster differ from nontelomeric retrotransposons in ways that give insight into general telomere biology. Here, we analyze telomere-specific retrotransposons from D. virilis, separated from D. melanogaster by 40 to 60 million years, to evaluate the evolutionary divergence of their telomeric traits. The telomeric retrotransposon HeT-A from D. melanogaster has an unusual promoter near its 3' terminus that drives not the element in which it resides, but the adjacent downstream element in a head-to-tail array. An obvious benefit of this promoter is that it adds nonessential sequence to the 5' end of each transcript, which is reverse transcribed and added to the chromosome. Because the 5' end of each newly transposed element forms the end of the chromosome until another element transposes onto it, this nonessential sequence can buffer erosion of sequence essential for HeT-A. Surprisingly, we have now found that HeT-A in D. virilis has a promoter typical of non-LTR retrotransposons. This promoter adds no buffering sequence; nevertheless, the complete 5' end of the element persists in telomere arrays, necessitating a more precise processing of the extreme end of the telomere in D. virilis. telomeres | retrotransposons | end replication problem | promoter evolution doi/ 10.1073/pnas.1000612107

Abscisic acid (ABA) is one of the most important phytohormones involved in abiotic stress responses, seed maturation, germination, and senescence. ABA is predominantly produced in vascular tissues and exerts hormonal responses in various cells, including guard cells. Although ABA responses require extrusion of ABA from ABA-producing cells in an intercellular ABA signaling pathway, the transport mechanisms of ABA through the plasma membrane remain unknown. Here we isolated an ATP-binding cassette (ABC) transporter gene, AtABCG25, from Arabidopsis by genetically screening for ABA sensitivity. AtABCG25 was expressed mainly in vascular tissues. The fluorescent protein-fused AtABCG25 was localized at the plasma membrane in plant cells. In membrane vesicles derived from AtABCG25-expressing insect cells, AtABCG25 exhibited ATP-dependent ABA transport. The AtABCG25-overexpressing plants showed higher leaf temperatures, implying an influence on stomatal regulation. These results strongly suggest that AtABCG25 is an exporter of ABA and is involved in the intercellular ABA signaling pathway. The presence of the ABA transport mechanism sheds light on the active control of multicellular ABA responses to environmental stresses among plant cells. Arabidopsis | ABCG | transposon-tagged lines doi/10.1073/pnas.0912516107

Here, we show that recombinant Drosophila elp1 (D-elp1) produced in Sf9 cells or Escherichia coil, corresponding to the largest of the three subunits in the RNA polymerase II core elongator complex, has RNA-dependent RNA polymerase (RdRP) activity. D-elp1 is a noncanonical RdRP that can synthesize dsRNA from different ssRNA templates using either a primer-dependent or primer-independent initiation mechanism. Of the three core subunits, only D-elp1 depletion inhibits RNAi in S2 cells but does not affect micro RNA function. Furthermore, D-elp1 depletion results in increased steady state levels of representative transposon RNAs and a decrease in the corresponding transposon antisense transcripts and endo siRNAs. In contrast, although Dcr-2 depletion results in increased transposon RNA levels and a reduction in the corresponding endo siRNAs, there is no change in the transposon antisense RNA levels. In D-elp1 null third instar larvae transposon RNA levels are also increased and the corresponding transposon antisense RNAs are reduced. D-elp1 associates tightly with Dcr-2, similar to the Dicer-RdRP interaction observed in lower eukaryotes. These results identify an aspect of the RNAi pathway in Drosophila that suggest transposon derived endo siRNAs, critical for transposon suppression, are produced, in part, in a D-elp1 dependent step that converts transposon RNA into dsRNA that is subsequently processed by Dcr-2. The generality of this mechanism in genome defense and RNA silencing in higher eukaryotes is suggested. elongator | genome defence | cellular stress

In fish, amino acids are food-related important olfactory cues to elicit an attractive response. However, the neural circuit underlying this olfactory behavior is not fully elucidated. In the present study, we applied the Tol2 transposon-mediated gene trap method to dissect the zebrafish olfactory system genetically. Four zebrafish lines (SAGFF27A, SAGFF91B, SAGFF179A, and SAGFF228C) were established in which the modified transcription activator Gal4FF was expressed in distinct subsets of olfactory sensory neurons (OSNs). The OSNs in individual lines projected axons to partially overlapping but mostly different glomeruli in the olfactory bulb (OB). In SAGFF27A, Gal4FF was expressed predominantly in microvillous OSNs innervating the lateral glomerular cluster that corresponded to the amino acid-responsive region in the OB. To clarify the olfactory neural pathway mediating the feeding behavior, we genetically expressed tetanus neurotoxin in the Gal4FF lines to block synaptic transmission in distinct populations of glomeruli and examined their behavioral response to amino acids. The attractive response to amino acids was abolished only in SAGFF27A fish carrying the tetanus neurotoxin transgene. These findings clearly demonstrate the functional significance of the microvillous OSNs innervating the lateral glomerular cluster in the amino acid-mediated feeding behavior of zebrafish. Thus, the integrated approach combining genetic, neuroanatomical, and behavioral methods enables us to elucidate the neural circuit mechanism underlying various olfactory behaviors in adult zebrafish. feeding behavior | Gal4/UAS | neural transmission blockade | olfaction

Horizontal transfer (HT) is central to the evolution of prokaryotic species. Selfish and mobile genetic elements, such as phages, plasmids, and transposons, are the primary vehicles for HT among prokaryotes. In multicellular eukaryotes, the prevalence and evolutionary significance of HT remain unclear. Here, we identified a set of DNA transposon families dubbed SPACE INVADERS (or SPIN) whose consensus sequences are [approximately equal to] 96% identical over their entire length (2.9 kb) in the genomes of murine rodents (rat/mouse), bushbaby (prosimian primate), little brown bat (laurasiatherian), tenrec (afrotherian), opossum (marsupial), and two non-mammalian tetrapods (anole lizard and African clawed frog). In contrast, SPIN elements were undetectable in other species represented in the sequence databases, including 19 other mammals with draft whole-genome assemblies. This patchy distribution, coupled with the extreme level of SPIN identity in widely divergent tetrapods and the overall lack of selective constraint acting on these elements, is incompatible with vertical inheritance, but strongly indicative of multiple horizontal introductions. We show that these germline infiltrations likely occurred around the same evolutionary time (15-46 mya) and spawned some of the largest bursts of DNA transposon activity ever recorded in any species lineage (nearly 100,000 SPIN copies per haploid genome in tenrec). The process also led to the emergence of a new gene in the murine lineage derived from a SPIN transposase. In summary, HT of DNA transposons has contributed significantly to shaping and diversifying the genomes of multiple mammalian and tetrapod species. genome evolution | lateral gene transfer | transposable elements | transposase

An active miniature inverted repeat transposable element (MITE), mPing, was discovered by computer-assisted analysis of rice genome sequence. The mPing element is mobile in rice cell culture and in a few rice strains where it has been amplified to > 1,000 copies during recent domestication. However, determination of the transposase source and characterization of the mechanism of transposition have been hampered by the high copy number of mPing and the presence of several candidate autonomous elements in the rice genome. Here, we report that mPing is active in Arabidopsis thaliana, where its transposition is catalyzed by three sources of transposase from rice: the autonomous Ping and Pong elements and by a cDNA derived from a Ping transcript. In addition to transposase, the product of a second element-encoded ORF of unknown function is also required for mPing transposition. Excision of mPing in A. thaliana is usually precise, and transposed copies usually insert into unlinked sites in the genome that are preferentially in or near genes. As such, this will be a valuable assay system for the dissection of MITE transposition and a potentially powerful tagging system for gene discovery in eukaryotes. autonomous element | PIF/Harbinger | transgenic

Mycoplasma genitalium has the smallest genome of any organism that can be grown in pure culture. It has a minimal metabolism and little genomic redundancy. Consequently, its genome is expected to be a close approximation to the minimal set of genes needed to sustain bacterial life. Using global transposon mutagenesis, we isolated and characterized gene disruption mutants for 100 different nonessential protein-coding genes. None of the 43 RNA-coding genes were disrupted. Herein, we identify 382 of the 482 M. genitalium protein-coding genes as essential, plus five sets of disrupted genes that encode proteins with potentially redundant essential functions, such as phosphate transport. Genes encoding proteins of unknown function constitute 28% of the essential protein-coding genes set. Disruption of some genes accelerated M. genitalium growth. minimal cell | Mycoplasma genitalium | synthetic biology | transposon mutagenesis

Mice carrying the lymphoproliferation mutation lpr are defective in T-cell development. The gene coding for the Fas antigen in normal and lpr mice were isolated and compared. The results showed that the Fas gene in lpr mice contains an early transposable element insertion at intron 2. This insertion causes premature transcriptional termination and aberrant splicing ofthe Fas antigen mRNA.

An isogenic line of Drosophila was subjected to heat shock treatment and the subsequent generation was analysed for transpositions of the copia-like Dm-412. Hybridization analysis showed the presence of multiple Dm-412 transpositions as a result of heat shock. The transpositions were nonrandom and occured at frequencies two-fold greater than those observed in control populations. The transposition events were dependent on the intensity of the stress rather than the stress type.

The presence of mouse embryonic stem (ES) cells makes the mouse a powerful model organism for reverse genetics, gene function study through mutagenesis of specific genes. In contrast, forward genetics, identification of mutated genes responsible for specific phenotypes, has an advantage to uncover novel pathways and unknown genes because no a priori assumptions are made about the mutated genes. However, it has been hampered in mice because of the lack of a system in which a large-scale mutagenesis and subsequent isolation of mutated genes can be performed efficiently. Here, we demonstrate the efficient chromosomal transposition of a Tc1/mariner-like transposon, Sleeping Beauty, in mice. This system allows germ-line mutagenesis in vivo and will facilitate certain aspects of phenotype-driven genetic screening in mice.