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Bacteroides are Gram-negative anaerobes indigenous to the intestinal tract of humans, and they are important opportunistic pathogens. Mobile genetic elements, such as conjugative transposons (CTns), have contributed to an increase in antibiotic resistance in these organisms. CTns are self-transmissible elements that belong to the superfamily of integrative and conjugative elements (ICEs). CTn341 is 52 kb; it encodes tetracycline resistance and its transfer is induced by tetracycline. The mobilization region of CTn341 was shown to be comprised of a three-gene operon, mobABC, and the transfer origin, oriT. The three genes code for a nicking accessory protein, a relaxase, and a VirD4-1ike coupling protein, respectively. The Mob proteins were predicted to mediate the formation of the relaxosome complex, nick DNA at the oriT, and shuttle the DNA/protein complex to the mating-pore apparatus. The results of mutational studies indicated that the three genes are required for maximal transfer of CTn341. Mob gene transcription was induced by tetracycline, and this regulation was mediated through the two-component regulatory system, RteAB. The oriT region of CTn341 was located within 100 bp of mobA, and a putative Bacteroides consensus nicking site was observed within this region. Mutation of the putative nick site resulted in a loss of transfer. This study demonstrated a role of the mobilization region for transfer of Bacteroides CTns and that tetracycline induction occurs for the mob gene operon, as for the tra gene operon(s), as shown previously. doi: 10.1128/JB.00317-10

Pigmentation patterning has long interested biologists, integrating topics in ecology, development, genetics, and physiology. Wildtype neonatal larvae of the silkworm, Bombyx mori, are completely black. By contrast, the epidermis and head of larvae of the homozygous recessive sex-linked chocolate (sch) mutant are reddish brown. When incubated at 30[degrees]C, mutants with the sch allele fail to hatch; moreover, homozygous mutants carrying the allele sch lethal (schI) do not hatch even at room temperature (25[degrees]C). By positional cloning, we narrowed a region containing sch to 239,622 bp on chromosome 1 using 4,501 backcross (BC1) individuals. Based on expression analyses, the best sch candidate gene was shown to be tyrosine hydroxylase (BmTh). BroTh coding sequences were identical among sch, schl, and wild-type. However, in sch the ~70-kb sequence was replaced with ~4.6 kb of a Tc1-mariner type transposon located ~6 kb upstream of BroTh, and in schl, a large fragment of an LIBm retrotransposon was inserted just in front of the transcription start site of BroTh. In both cases, we observed a drastic reduction of BroTh expression. Use of RNAi with BroTh prevented pigmentation and hatching, and feeding of a tyrosine hydroxylase inhibitor also suppressed larval pigmentation in the wild-type strain, [pnd.sup.+] and in a pS (black-striped) heterozygote. Feeding L-dopa to sch neonate larvae rescued the mutant phenotype from chocolate to black. Our results indicate the BrnTh gene is responsible for the sch mutation, which plays an important role in melanin synthesis producing neonatal larval color. doi/ 10.1073/pnas.1001725107

The western clawed frog Xenopus tropicolis is an important model for vertebrate development that combines experimental advantages of the African clawed frog Xenopus laevis with more tractable genetics. Here we present a draft genorne sequence assembly of X. tropicalis. This genome encodes more than 20,000 protein-coding genes, including orthologs of at least 1700 human disease genes. Over 1 million expressed sequence tags validated the annotation. More than one-third of the genome consists of transposable elements, with unusually prevalent DNA transposons. Like that of other tetrapods, the genome of X. tropicolis contains gene deserts enriched for conserved noncoding elements. The genome exhibits substantial shared synteny with human and chicken over major parts of large chromosomes, broken by lineage-specific chromosome fusions and fissions, mainly in the mammalian lineage. doi: 10.1126/science.1183670

Transposable elements, particularly LTR-retrotransposons, comprise the primary vehicle for genome size expansion in plants, while DNA removal through illegitimate recombination and intrastrand homologous recombination serve as the most important counteracting forces to plant genomic obesity. Despite extensive research, the relative impact of these opposing forces and hence the directionality of genome size change remains unknown. In Gossypium (cotton), the 3-fold genome size variation among diploids is due largely to copy number variation of the gypsy-like retrotransposon Gorge3. Here we combine comparative sequence analysis with a modeling approach to study the directionality of genome size change in Gossypium. We demonstrate that the rate of DNA removal in the smaller genomes is sufficient to reverse genome expansion through Gorge3 proliferation. These data indicate that rates of DNA loss can be highly variable even within a single plant genus, and that the known mechanisms of DNA loss can indeed reverse the march toward genomic obesity. C-value I cotton | genome evolution | Gossypium | transposable elements doi/ 10.1073/pnas.0904339106

Miniature inverted repeat transposable elements (MITEs) are widespread in eukaryotic genomes, where they can attain high copy numbers despite a Lack of coding capacity. However, little is known about how they originate and amplify. We performed a genome-wide screen of functional interactions between Stowoway MITEs and potential transposases in the rice genome and identified a transpositionally active MITE that possesses key properties that enhance transposition. Although not directly related to its autonomous element, the MITE has Less affinity for the transposase than does the autonomous element but lacks a motif repressing transposition in the autonomous element. The MITE contains internal sequences that enhance transposition. These findings suggest that MITEs achieve high transposition activity by scavenging transposases encoded by distantly related and self-restrained autonomous elements.

We identified Xenorhabdus nematophila transposon mutants with defects in lipase activity. One of the mutations, in yigL, a conserved gene of unknown function, resulted in attenuated virulence against Manduca sexta insects. We discuss possible connections between lipase production, YigL, and specific metabolic pathways.

Whole genome sequencing of the model organisms has created increased demand for efficient tools to facilitate the genome annotation efforts. Accordingly, we report the further implementations and analyses stemming from our publicly available P[wHy] library for Drosophila melanogaster. A two-step regime--large scale transposon mutagenesis followed by hobo-induced nested deletions--allows mutation saturation and provides significant enhancements to existing genomic coverage. We previously showed that, for a given starting insert, deletion saturation is readily obtained over a 60-kb interval; here, we perform a breakdown analysis of efficiency to identify rate-limiting steps in the process. Transrecombination, the hobo-induced recombination between two P[wHy] half molecules, was shown to further expand the P[wHy] mutational range, pointing to a potent, iterative process of transrecombination-reconstitution-transrecombination for alternating between very large and very fine-grained deletions in a self-contained manner. A number of strains also showed partial or complete repression of P[wHy] markers, depending on chromosome location, whereby asymmetric marker silencing allowed continuous phenotypic detection, indicating that P[wHy]-based saturational mutagenesis should be useful for the study of heterochromatin/positional effects. DOI: 10.1534/genetics.109.103762

Structured inquiry approaches, in which students receive a Drosophila strain of unknown genotype to analyze and map the constituent mutations, are a common feature of many genetics teaching laboratories. The required crosses frustrate many students because they are aware that they are participating in a fundamentally trivial exercise, as the map locations of the genes are already established and have been recalculated thousands of times by generations of students. We modified the traditional structured inquiry approach to include a novel research experience for the students in our undergraduate genetics laboratories. Students conducted crosses with Drosophila strains carrying P[lacW] transposon insertions in genes without documented recombination map positions, representing a large number of unique, but equivalent genetic unknowns. Using the eye color phenotypes associated with the inserts as visible markers, it is straightforward to calculate recombination map positions for the interrupted loci. Collectively, our students mapped 95 genetic loci on chromosomes 2 and 3. In most cases, the calculated 95% confidence interval for meiotic map location overlapped with the predicted map position based on cytology. The research experience evoked positive student responses and helped students better understand the nature of scientific research for little additional cost or instructor effort. DOI: 10.1534/genetics. 109.101774

Galileo is the only transposable element (TE) known to have generated natural chromosomal inversions in the genus Drosophila. It was discovered in Drosophila buzzatii and classified as a Foldback-like element because of its long, internally repetitive, terminal inverted repeats (TIRs) and lack of coding capacity. Here, we characterized a seemingly complete copy of Galileo from the D. buzzatii genome. It is 5,406 bp long, possesses 1,229-bp TIRs, and encodes a 912-aa transposase similar to those of the Drosophila melanogaster 1360 (Hoppel) and P elements. We also searched therecently available genome sequences of 12 Drosophila species for elements similar to Dbuz\Galileo by using bioinformatic tools. Galileo was found in six species (ananassae, willistoni, peudoobscura, persimilis, virilis, and mojavensis) from the two main lineages within the Drosophila genus. Our observations place Galileo within the P superfamily of cut-and-paste transposons and extend considerably its phylogenetic distribution. The interspecific distribution of Galileo indicates an ancient presence in the genus, but the phylogenetic tree built with the transposase amino acid sequences contrasts significantly with that of the species, indicating lineage sorting and/or horizontal transfer events. Our results also suggest that Foldback-like elements such as Galileo may evolve from DNA-based transposon ancestors by loss of the transposase gene and disproportionate elongation of TIRs. class II elements | transposase | terminal inverted repeats | 1360 | inversions

Abstract: An efficient system was developed, and several variables tested, for generating a large-scale insertionalmutagenesis population of rice. The most important feature in this improved Ac/Ds tagging system is that [...]

The transposon MTnSag1 from Streptococcus agalactiae carried an IS1-like transposase gene and the lnu(C) gene, which encoded a lincosamide nucleotidyltransferase. MTnSag1 could be mobilized by the conjugative transposon Tn916. An intermediate circular form of MTnSag1 and a putative origin of transfer at the 3' end of the lnu(C) gene were characterized.

Pseudomonas veronii MEK700 was isolated from a biotrickling filter cleaning 2-butanone-loaded waste air. The strain is able to grow on 2-butanone and 2-hexanol. The genes for degradation of short chain alkyl methyl ketones were identified by transposon mutagenesis using a newly designed transposon, mini-Tn5495, and cloned in Escherichia coli. DNA sequence analysis of a 15-kb fragment revealed three genes involved in methyl ketone degradation. The deduced amino acid sequence of the first gene, mekA, had high similarity to Baeyer-Villiger monooxygenases; the protein of the second gene, mekB, had similarity to homoserine acetyltransferases; the third gene, mekR, encoded a putative transcriptional activator of the AraC/XylS family. The three genes were located between two gene groups: one comprising a putative phosphoenolpyruvate synthase and glycogen synthase, and the other eight genes for the subunits of an ATPase. Inactivation of mekA and mekB by insertion of the mini-transposon abolished growth of P. veronii MEK700 on 2-butanone and 2-hexanol. The involvement of mekR in methyl ketone degradation was observed by heterologous expression of mekA and mekB in Pseudomonas putida. A fragment containing mekA and mekB on a plasmid was not sufficient to allow P. putida KT2440 to grow on 2-butanone. Not until all three genes were assembled in the recombinant P. putida was it able to use 2-butanone as carbon source. The Baeyer-Villiger monooxygenase activity of MekA was clearly demonstrated by incubating a mekB transposon insertion mutant of P. veronii with 2-butanone. Hereby, ethyl acetate was accumulated. To our knowledge, this is the first time that ethyl acetate by gas chromatographic analysis has been definitely demonstrated to be an intermediate of MEK degradation. The mekB-encoded protein was heterologously expressed in E. coli and purified by immobilized metal affinity chromatography. The protein exhibited high esterase activity towards short chain esters like ethyl acetate and 4-nitrophenyl acetate. doi:10.1128/JB.01279-06

A transposon site hybridization (TraSH) assay was developed for functional analysis of the Bacillus anthracis genome using a mini-Tnl0 transposon which permitted analysis of 82% of this pathogen's genes. The system, used to identify genes required for generation of infectious anthrax spores, spore germination, and optimal growth on rich medium, was predictive of the contributions of two conserved hypothetical genes for the phenotypes examined.

A type II transposon not previously reported in basidiomycetes has been discovered in Agaricus bisporus. Southern blot analysis shows little variation among commercial species, but great variation among mushrooms collected in the wild.

An investigation was conducted to isolate the gene responsible for Huntington's disease (HD). A direct cDNA selection strategy was used to narrow down the region of interest, resulting in the identification of seven clones within this region. One of the clones detected an insertion of 331 base pairs in two HD patients. Sequence analysis showed that the insertion element is a member of the Alu family and lies in close proximity to the alpha-adducin gene and another gene encoding a 12-kb transcript. Since the Alu insertion is not likely to affect the expression of the alpha-adducin gene, the second gene is a strong candidate forthe HD gene.

Vancomycin resistance in Enterococcus faecium is mediated by the van genes, of which one, vanA, is responsible for the synthesis of depsipeptides that can be incorporated into peptidoglycan precursors. The flanking regions of the van gene cluster in pIP816 were characterized. The results showed that the cluster is contained within a 10,851-bp transposon, designated Tn1546. Further characterization of this transposon showed that it belongs to the Tn3 family of transposons and is present in other enterococci, suggesting that transposition is a major mechanism of van gene cluster dissemination in enterococci.

The cloning from the Australian sheep blowfly (Luciliacuprina) of two DNA elements similar to the transposable P element of Drosophila melanogaster was reported. The D. melanogaster P element is an important tool in studies involving genetic manipulation, mutagenesis and germ-line transformation. The DNA elements from the blowfly were named Lu-P1 and Lu-P2. Their discovery outside of the family Drosophilidae indicate that transposons may be more widespread than previously believed.

The vasotocin gene of the Pacific hagfish Eptatretus stouti was cloned and characterized. Sequence analysis showed that the hagfish vasoticin gene contains an additional intron in the 5' untranslated region. Further analysis of the noncoding strand of this third intron showed the presence of a coding region for a Tc1-like element. Tc1 has previously been described in nematodes and Drosophila. These results indicate that this transposon family has a wider distribution than previously determined.

All eukaryotic DNA transposons reported so far belong to a single category of elements transposed by the so-called 'cut-and-paste' mechanism. Here, we report a previously unknown category of eukaryotic DNA transposons, Helitron, which transpose by rolling-circle replication. Autonomous Helitrons encode a 5'-to-3' DNA helicase and nuclease/ligase similar to those encoded by known rolling-circle replicons. Helitron-like transposons have conservative 5'-TC and CTRR-3' termini and do not have terminal inverted repeats. They contain 16- to 20-bp hairpins separated by 10-12 nucleotides from the 3'-end and transpose precisely between the 5'-A and T-3', with no modifications of the AT target sites. Together with their multiple diverged nonautonomous descendants, Helitrons constitute [approximately equals] 2% of both the Arabidopsis thaliana and Caenorhabditis elegans genomes and also colonize the Oriza sativa genome. Sequence conservation suggests that Helitrons continue to be transposed.

Some recent advances in the study of mammalian transposition are discussed. Such studies are hampered by the absence of an appropriate model system, and has depended mostly on experiments conducted on yeast and Drosophila genomes. One promising avenue is in the use of advanced glycosylation end products (AGE) to induce DNA damage in mammalian cells. Experiments using AGE have identified a sequence termed as INS-1, which may represent a unique type of mammalian transposing element.