Ion-exchange chromatography followed by ESI-MS for quantitative analysis of sugar monophosphates from glucose catabolism.

The aim of this work is to establish a quantitative method to determine the ratio of [U-(13)C] labeled to unlabeled hexose monophosphates isolated from yeast extracts. This is accomplished by anion exchange chromatography and mobile phase desalting followed by electrospray (ESI) mass spectrometry. We test the method with the analysis of a sample of biological origin.

Regulation of T-cell receptor beta-chain gene assembly by recombination signals: the beyond 12/23 restriction.

Assembly of antigen receptor genes is regulated in several important contexts during lymphocyte development. This regulation occurs through modulation of gene segment accessibility to the V(D)J recombinase and/or at the level of the recombination reaction due, in part, to constraints imposed by recombination signal (RS) sequences. RSs are composed of conserved heptamer and nonamer sequences that flank relatively non-conserved spacer sequences of either 12 or 23 base pairs.

The B12/23 restriction is critically dependent on recombination signal nonamer and spacer sequences.

Ag receptor variable region gene assembly is initiated through the formation of a synaptic complex which minimally includes the recombination-activating gene (RAG) 1/2 proteins and a pair of recombination signals (RSs) flanking the recombining gene segments. RSs are composed of conserved heptamer and nonamer sequences flanking relatively nonconserved spacers of 12 or 23 bp. RSs regulate variable region gene assembly within the context of the 12/23 rule which mandates that recombination only occurs between RSs of dissimilar spacer length.

T cell receptor CDR3 loop length repertoire is determined primarily by features of the V(D)J recombination reaction.

The third complementarity-determining region (CDR) of the TCR alpha and beta chains forms loops that engage amino acid residues of peptides complexed with MHC. This interaction is central to the specific discrimination of antigenic-peptide-MHC complexes by the TCR. The TCRbeta chain CDR3 loop is encoded by the Dbeta gene segment and flanking portions of the Vbeta and Jbeta gene segments.

Restrictions limiting the generation of DNA double strand breaks during chromosomal V(D)J recombination.

Antigen receptor loci are composed of numerous variable (V), diversity (D), and joining (J) gene segments, each flanked by recombination signal sequences (RSSs). The V(D)J recombination reaction proceeds through RSS recognition and DNA cleavage steps making it possible for multiple DNA double strand breaks (DSBs) to be introduced at a single locus. Here we use ligation-mediated PCR to analyze DNA cleavage intermediates in thymocytes from mice with targeted RSS mutations at the endogenous TCRbeta locus.