Targeted transposition by the V(D)J recombinase.

Cleavage by the V(D)J recombinase at a pair of recombination signal sequences creates two coding ends and two signal ends. The RAG proteins can integrate these signal ends, without sequence specificity, into an unrelated target DNA molecule. Here we demonstrate that such transposition events are greatly stimulated by--and specifically targeted to--hairpins and other distorted DNA structures. The mechanism of target selection by the RAG proteins thus appears to involve recognition of distorted DNA. These data also suggest a novel mechanism for the formation of alternative recombination products termed hybrid joints, in which a signal end is joined to a hairpin coding end. We suggest that hybrid joints may arise by transposition in vivo and propose a new model to account for some recurrent chromosome translocations found in human lymphomas. According to this model, transposition can join antigen receptor loci to partner sites that lack recombination signal sequence elements but bear particular structural features. The RAG proteins are capable of mediating all necessary breakage and joining events on both partner chromosomes; thus, the V(D)J recombinase may be far more culpable for oncogenic translocations than has been suspected.

A means to accommodate residual limb movement during optical scanning: a technical note.

A technique is described for correcting for subject movement while imaging the residual limb of a person with a transtibial amputation. Small reflective markers were placed on the residual limb, and then their motions tracked during scanning using two stationary cameras. The marker position measurements were used to generate appropriate translational and rotational transformation matrices so that limb motion could be corrected for during the 1.5-s scan interval. Evaluation tests showed good performance for moderate (2-4 mm) to high (5-8 mm) motion cases. The difference in mean absolute cross-sectional area between the test scan and a stationary reference scan was reduced by approximately one half when motion correction was used compared with when motion correction was not used. The algorithm broke down for exaggerated motion ( >or= 9 mm) cases, particularly in areas outside the region encompassed by the markers. The developed method is useful in prosthetics research where high resolution shape measurement is needed, for example in cases where residual limb shape or volume change is of interest.

CAD/CAM transtibial prosthetic sockets from central fabrication facilities: how accurate are they?

This research compares transtibial prosthetic sockets made by central fabrication facilities with their corresponding American Academy of Orthotists and Prosthetists (AAOP) electronic shape files and assesses the central fabrication process. We ordered three different socket shapes from each of 10 manufacturers. Then we digitized the sockets using a very accurate custom mechanical digitizer. Results showed that quality varied considerably among the different manufacturers. Four of the companies consistently made sockets within +/-1.1% volume (approximately 1 sock ply) of the AAOP electronic shape file, while six other companies did not. Six of the companies showed consistent undersizing or oversizing in their sockets, which suggests a consistent calibration or manufacturing error. Other companies showed inconsistent sizing or shape distortion, a difficult problem that represents a most challenging limitation for central fabrication facilities.

B cell development leads off with a base hit: dU:dG mismatches in class switching and hypermutation.

The mechanisms underlying somatic hypermutation (SHM) and class switch recombination (CSR) have been the subject of much debate. Recent studies from the Neuberger and Honjo labs have lent insight into these distinct processes, and we discuss a new, comprehensive model for how AID, uracil DNA glycosylase (UNG) and the mismatch repair system function in both SHM and CSR.

RAG proteins shepherd double-strand breaks to a specific pathway, suppressing error-prone repair, but RAG nicking initiates homologous recombination.

The two major pathways for repairing double-strand breaks (DSBs), homologous recombination and nonhomologous end joining (NHEJ), have traditionally been thought to operate in different stages of the cell cycle. This division of labor is not absolute, however, and precisely what governs the choice of pathway to repair a given DSB has remained enigmatic. We pursued this question by studying the site-specific DSBs created during V(D)J recombination, which relies on classical NHEJ to repair the broken ends. We show that mutations that form unstable RAG postcleavage complexes allow DNA ends to participate in both homologous recombination and the error-prone alternative NHEJ pathway. By abrogating a key function of the complex, these mutations reveal it to be a molecular shepherd that guides DSBs to the proper pathway. We also find that RAG-mediated nicks efficiently stimulate homologous recombination and discuss the implications of these findings for oncogenic chromosomal rearrangements, evolution, and gene targeting.

The V(D)J recombinase efficiently cleaves and transposes signal joints.

V(D)J recombination generates two types of products: coding joints, which constitute the rearranged variable regions of antigen receptor genes, and signal joints, which often form on immunologically irrelevant, excised circular molecules that are lost during cell division. It has been widely believed that signal joints simply convert reactive broken DNA ends into safe, inert products. Yet two curious in vivo observations made us question this assumption: signal ends are far more abundant than coding ends, and signal joints form only after RAG expression is downregulated. In fact, we find that signal joints are not at all inert; they are cleaved quite efficiently in vivo and in vitro by a nick-nick mechanism and form an excellent substrate for RAG-mediated transposition in vitro, possibly explaining how genomic stability in lymphocytes may be compromised.