SUMMARY Hermes is a member of the hAT transposon superfamily which has active representatives, including McClintock's archetypal Ac mobile genetic element, in many eukaryotic species. The crystal structure of the Hermes transposase-DNA complex reveals that Hermes forms an octameric ring organized as a tetramer of dimers. While isolated dimers are active in vitro for all the chemical steps of transposition, only octamers are active in vivo. The octamer can provide not only multiple specific DNA-binding domains to recognize repeated subterminal sequences within the transposon ends, which are important for activity, but also multiple non-specific DNA binding surfaces for target capture. The unusual assembly explains the basis of bipartite DNA recognition at hAT transposon ends, provides a rationale for transposon end asymmetry, and suggests how the avidity provided by multiple sites of interaction could allow a transposase to locate its transposon ends amidst a sea of chromosomal DNA.

The carboxylesterase Est55 has been cloned and expressed in Bacillus subtilis strains. Est55, which lacks a classical, cleavable N-terminal signal sequence, was found to be secreted during the stationary phase of growth such that there is more Est55 in the medium than inside the cells. Several cytoplasmic proteins were also secreted in large amounts during late stationary phase, indicating that secretion in B. subtilis is not unique to Est55. These proteins, which all have defined cytoplasmic functions, include GroEL, DnaK, enolase, pyruvate dehydrogenase subunits PdhB and PdhD, and SodA. The release of Est55 and those proteins into the growth medium is not due to gross cell lysis, a conclusion that is supported by several lines of evidence: constant cell density and secretion in the presence of chloramphenicol, constant viability count, the absence of EF-Tu and SecA in the culture medium, and the lack of effect of autolysin-deficient mutants. The shedding of these proteins by membrane vesicles into the medium is minimal. More importantly, we have identified a hydrophobic ␣-helical domain within enolase that contributes to its secretion. Thus, upon the genetic deletion or replacement of a potential membrane-embedding domain, the secretion of plasmid gene-encoded mutant enolase is totally blocked, while the wild-type chromosomal enolase is secreted normally in the same cultures during the stationary phase, indicating differential specificity. We conclude that the secretion of Est55 and several cytoplasmic proteins without signal peptides in B. subtilis is a general phenomenon and is not a consequence of cell lysis or membrane shedding; instead, their secretion is through a process(es) in which protein domain structure plays a contributing factor.Bacillus subtilis secretes large amounts of proteins into the growth medium (43). Of the known secretory pathways in B. subtilis, the majority of proteins are exported from the cytoplasm by the Sec-dependent pathway, through which secretory proteins are synthesized as precursors with typical cleavable N-terminal signal peptides (3, 36). Fewer proteins are released into the medium via the cleavable twin-arginine translocation (TAT) system (39). Still other proteins are exported into the medium via ATP-binding cassette transporters, a dedicated pseudopilin export pathway, a competence development system or an ESAT-6 (Mycobacterium tuberculosis early secreted antigenic target of 6 kDa)-like system (31).The genome of B. subtilis 168 is 4,215 kbp in length and contains about 4,100 genes that are predicted to include over 250 extracellular proteins; the majority of these proteins are secreted through the aforementioned pathways (3, 18). However, proteomic studies have revealed that genome-based predictions reflect only 50% of the actual composition of the extracellular proteome. This significant discrepancy is mainly due to the difficulties in the prediction of extracellular proteins lacking signal peptides (including cytoplasmic proteins) and lipoproteins (3, 18). These findin...

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Chromosome structure and function are influenced by transposable elements, which are mobile DNA segments that can move from place to place. hAT elements are a superfamily of DNA cut and paste elements that move by excision and integration. We have characterized two hAT elements, TcBuster and Space Invaders (SPIN), that are members of a recently described subfamily of hAT elements called Buster elements. We show that TcBuster, from the red flour beetle Tribolium castaneum, is highly active in human cells. SPIN elements are currently inactive elements that were recently highly active in multiple vertebrate genomes, and the high level of sequence similarity across widely diverged species and patchy phylogenetic distribution suggest that they may have moved between genomes by horizontal transfer. We have generated an intact version of this element, SPIN ON , which is highly active in human cells. In vitro analysis of TcBuster and SPIN ON shows that no proteins other than transposase are essential for recombination, a property that may contribute to the ability of SPIN to successfully invade multiple organisms. We also analyze the target site preferences of de novo insertions in the human genome of TcBuster and SPIN ON and compare them with the preferences of Sleeping Beauty and piggyBac, showing that each superfamily has a distinctive pattern of insertion. The high-frequency transposition of both TcBuster and SPIN ON suggests that these transposon systems offer powerful tools for genome engineering. Finally, we describe a Saccharomyces cerevisiae assay for TcBuster that will provide a means for isolation of hyperactive and other interesting classes of transposase mutants.hAT element | target site selection | gene therapy | insertional mutagenesis | transgenesis