Productive gene rearrangement at the T-cell receptor (TCR) beta-chain locus facilitates formation of the "pre-TCR," a molecular complex that is important for the subsequent development of alpha beta T cells. The transition of thymocytes from a population of cells undergoing TCRbeta chain genes to a population enriched in cells with productively rearranged TCRbeta chain genes is known as "beta selection." This is the first point in alpha beta T-cell development at which the products of an activated TCR locus define cell phenotype. Toward an understanding of these events, this study has focused on a set of thymocytes defined by cell surface phenotype as HSA+ CD44low CD25+, in which the bulk of TCRbeta gene rearrangement occurs. The analysis of this set, presented here, allows its novel subdivision into two subsets that are respectively strong candidates for cells immediately prior to and immediately following TCRbeta selection. Cells that have passed beta selection differ from the preceding cells by several criteria, including hyperphosphorylation of Rb, increased expression of cyclins A and B, down-regulation of p27, increased CDK2 activity, an induction of cdc2 activity, and progression through DNA synthesis. Consistent with these changes being attributable to productive TCRbeta chain gene rearrangement, the identified "beta-selected" subset is not detected in mutant mice that cannot assemble a pre-TCR. Interestingly, there is a coincident selective and transient down-regulation of the protein RAG2, on which TCR gene rearrangement obligatorily depends. Together, these findings demonstrate that productive TCR gene rearrangement is associated with events that can ensure thymocyte expansion and monoclonality.

Two waves of immunoglobulin gene rearrangements, first of the heavy, then of the light chain chain gene loci form functional immunoglobulin genes during B cell development. In mouse bone marrow the differential surface expression of B220 (CD45R), c-kit, CD25, and surrogate light chain as well as the cell cycle status allows FACS separation of the cells in which these two waves of rearrangements occur. The gene products of two recombination activating genes, RAG1 and RAG2 are crucial for this rearrangement process. Here, we show that the expression of the RAG genes is twice up- and down-regulated, at the transcriptional level for RAG1 and RAG2, and at the postranscriptional level for RAG2 protein. Expression levels are high in D-->JH and VH-->DJH rearranging proB and preB-I cells, low in preB cells expressing the preB cell receptor on the cell surface, and high again in VL-->JL rearranging small preB-II cells. In immature B cells expressing on the cell surface RAG1 and RAG2 mRNA is down-regulated, whereas RAG2 protein levels are maintained. Down-regulation of RAG1 and RAG2 gene expression after productive rearrangement at one heavy chain allele might be part of the mechanisms that prevent further rearrangements at the other allele.

V(D)J recombination, the process that assembles antigen-receptor genes, is directed by signal sequences flanking the DNA segments to be joined. Signals consist of a conserved heptamer and nonamer separated by a spacer of either 12 or 23 base pairs. Recombination occurs almost exclusively between two signals with spacers of different lengths. This restriction, called the '12/23 rule', governs the organization and pattern of rearrangement of antigen-receptor loci. In vitro work demonstrating the direct roles of the Rag proteins in the initiation of V(D)J recombination did not recreate the 12/23 rule. Instead, double-strand breaks were formed efficiently at isolated signals. Here we show that extracts made from a lymphoid cell line that expresses truncated forms of the Rag1 and Rag2 proteins have a signal-cutting activity that obeys the 12/23 rule. Cleavage at the two signals is concerted and requires their synapsis, and mutations of one signal prevent cleavage at both.

Stress generated by humans on wildlife by continuous development of outdoor recreational activities is of increasing concern for biodiversity conservation. Human disturbance often adds to other negative impact factors affecting the dynamics of vulnerable populations. It is not known to which extent the rapidly spreading free-riding snow sports actually elicit detrimental stress (allostatic overload) upon wildlife, nor what the potential associated fitness and survival costs are. Using a non-invasive technique, we evaluated the physiological stress response induced by free-riding snow sports on a declining bird species of Alpine ecosystems. The results of a field experiment in which radiomonitored black grouse (Tetrao tetrix) were actively flushed from their snow burrows once a day during four consecutive days showed an increase in the concentration of faecal stress hormone (corticosterone) metabolites after disturbance. A large-scale comparative analysis across the southwestern Swiss Alps indicated that birds had higher levels of these metabolites in human-disturbed versus undisturbed habitats. Disturbance by snow sport free-riders appears to elevate stress, which potentially represents a new serious threat for wildlife. The fitness and survival costs of allostatic adjustments have yet to be estimated.

Despite the essential and synergistic functions of the rag-1 and rag-2 proteins in V(D)J recombination and lymphocyte development, little is known about the biochemical properties of the two proteins. We have developed cell lines expressing high levels of the rag proteins and specific, sensitive immunological reagents for their detection, and we have examined the physical properties of the rag proteins in vitro and their subcellular localizations in vivo. rag-1 is tightly associated with nuclear structures, requires a high salt concentration to maintain its solubility, and is a component of large, heterogeneously sized complexes. Furthermore, the presence of rag-1 alters the behavior of rag-2, conferring on it properties similar to those of rag-1 and changing its distribution in the nucleus. We demonstrate that rag-1 and rag-2 are present in the same complex by coimmunoprecipitation, and we provide evidence that these complexes contain more molecules of rag-2 than of rag-1. The demonstration of intracellular complexes containing rag-1 and rag-2 raises the possibility that interaction between these proteins is necessary for their biological function.