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T M Leu

Publications and source records attributed to T M Leu.

10 recordsLinked to original sources
PubMed1997

Coding joint formation in a cell-free V(D)J recombination system.

V(D)J recombination assembles the variable portion of antigen receptor genes in developing lymphocytes and is the only site-specific recombination reaction known in vertebrates. A cell-free system has been established that performs DNA cleavage, end processing, and joining to yield V(D)J coding joints that exhibit structural features similar to those formed in vivo. The reaction has the expected substrate, metal ion, and RAG protein requirements. The efficiency of coding joint formation is reduced dramatically by uncoupling the cleavage and joining portions of the reaction, indicating that a postcleavage coding end complex facilitates joining. By varying the reaction conditions, nucleotide loss from coding ends and heterogeneity of coding joints can be regulated. This cell-free system provides a novel tool for detailed mechanistic analyses of the end processing and joining steps of V(D)J recombination.

Cell-Free System↗
PubMed1996

Productive T-cell receptor beta-chain gene rearrangement: coincident regulation of cell cycle and clonality during development in vivo.

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.

Animals↗
PubMed1996

The half-life of RAG-1 protein in precursor B cells is increased in the absence of RAG-2 expression.

Site-specific recombination of immunoglobulin and T cell receptor gene segments in B and T lymphocytes is dependent on the expression of two recombinant activation genes, Rag-1 and Rag-2. Here, we show that RAG-1 protein turnover in pre-B cells depends on the expression of RAG-2. The apparent half-life of RAG-1 protein is increased when RAG-2 is not expressed in differentiating pre-B cells.

Animals↗
PubMed1996

Initiation of V(D)J recombination in vitro obeying the 12/23 rule.

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.

Base Sequence↗
PubMed1995

Down-regulation of RAG1 and RAG2 gene expression in preB cells after functional immunoglobulin heavy chain rearrangement.

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.

Animals↗
PubMed1995

rag-1 and rag-2 are components of a high-molecular-weight complex, and association of rag-2 with this complex is rag-1 dependent.

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.

Animals↗
PubMed1992

Anticardiolipin antibody in stroke.

Anticardiolipin antibody (ACA) has been associated with cerebral ischemia, suggesting an important role in the pathogenesis of vascular thrombosis and a marker for increased thrombotic risk. Its prevalence and significance in stroke are unknown. In this study, consecutively admitted patients diagnosed as having stroke were studied. A total of 246 patients, 141 men and 105 women, aged 34 to 91 years (mean age, 63.5 years), were screened for the presence of ACA. Elevated concentration of circulating ACA was present in 4 out of 170 patients with infarct, and in 1 out of 76 patients with hemorrhage. They were 4 men and 1 woman, aged 49 to 84 years (mean age, 66.8 years). The prevalence of ACA in stroke was 2% (2.3% for infarction and 1.3% for hemorrhage). All the elevated ACA were of IgG isotype. No strong association between antibody positivity and stroke was found in this study. The routine screen of ACA in stroke is of questionable value.

Adult↗
PubMed1992

Bilateral putaminal necrosis caused by methanol poisoning: a case report.

Bilateral putaminal necrosis is characteristic of methanol poisoning. A 31-year-old male alcoholic had headache, impaired consciousness, neck stiffness, roving eyes with dilated unreactive pupils, papilloedema, abdominal pain, vomiting, and severe metabolic acidosis after a binge. Abnormalities of the cerebrospinal fluid included an initial pressure of 240 mmH2 O, RBC 286/mm3, WBC 8/mm3, and protein 179 mg/dl. Peritoneal dialysis was performed on the 2nd day after drinking. A blood test for methanol was not performed until the 5th day, and its results was negative. However, computed tomography (CT) on the 3rd day showed necrosis and hemorrhage of bilateral putamina and the cerebral cortex, and post-contrast enhancement of meninges. On the 22nd day, a CT revealed further changes: necrosis of bilateral subcortical white matter, and post-contrast gyral enhancement at the otherwise normal-looking areas of the cerebral cortex. We suggest that, in certain situations, the characteristic CT findings are helpful in the diagnosis of methanol poisoning.

Adult↗