subsp. Candidate Vaccine Strains Are Pro-apoptotic in RAW 264.7 Murine Macrophages.

subsp. (MAP) is the etiological agent of Johne's disease, a severe gastroenteritis of ruminants. This study developed a model cell culture system to rapidly screen MAP mutants with vaccine potential for apoptosis.

Isolation of Plant Nuclei Compatible with Microfluidic Single-nucleus ATAC-sequencing.

Gene expression depends on the binding of transcription factors with DNA regulatory sequences. The level of accessibility for these sequences varies between cells and cell types. Until recently, using the Tn5 assay for transposase-accessible chromatin for sequencing (ATAC-seq) technology allowed assessing the profiles of chromatin from an entire organ or, when coupled with the isolation of nuclei tagged in specific cell types (INTACT) method, from a cell-type.

Accelerated progression of chronic lymphocytic leukemia in Eμ-TCL1 mice expressing catalytically inactive RAG1.

Chronic lymphocytic leukemia (CLL) is a prevalent B-cell neoplasia that is often preceded by a more benign monoclonal CD5(+) B-cell lymphocytosis. We previously generated transgenic mice expressing catalytically inactive RAG1 (dominant-negative recombination activating gene 1 [dnRAG1] mice) that develop an early-onset indolent CD5(+) B-cell lymphocytosis attributed to a defect in secondary V(D)J rearrangements initiated to edit autoreactive B-cell receptor (BCR) specificity. Hypothesizing that CD5(+) B cells in these animals represent potential CLL precursors, we crossed dnRAG1 mice with CLL-prone Eμ-TCL1 mice to determine whether dnRAG1 expression in Eμ-TCL1 mice accelerates CLL onset.

VprBP binds full-length RAG1 and is required for B-cell development and V(D)J recombination fidelity.

The N-terminus of full-length RAG1, though dispensable for RAG1/2 cleavage activity, is required for efficient V(D)J recombination. This region supports RING E3 ubiquitin ligase activity in vitro, but whether full-length RAG1 functions as a single subunit or a multi-subunit E3 ligase in vivo is unclear. We show the multi-subunit cullin RING E3 ligase complex VprBP/DDB1/Cul4A/Roc1 associates with full-length RAG1 through VprBP.

Accumulation of B1-like B cells in transgenic mice over-expressing catalytically inactive RAG1 in the periphery.

During their development, B lymphocytes undergo V(D)J recombination events and selection processes that, if successfully completed, produce mature B cells expressing a non-self-reactive B-cell receptor (BCR). Primary V(D)J rearrangements yield self-reactive B cells at high frequency, triggering attempts to remove, silence, or reprogramme them through deletion, anergy induction, or secondary V(D)J recombination (receptor editing), respectively. In principle, expressing a catalytically inactive V(D)J recombinase during a developmental stage in which V(D)J rearrangement is initiated may impair this process.

Cigarette smoke-induced effects on bone marrow B-cell subsets and CD4+:CD8+ T-cell ratios are reversed by smoking cessation: influence of bone mass on immune cell response to and recovery from smoke exposure.

Cigarette smoking adversely affects the immune system, and is a risk factor for developing osteoporosis. How smoking contributes to osteoporosis is unclear, but since lymphocytes help maintain bone homeostasis and lymphocyte depletion results in bone loss, one potential mechanism for how smoke exposure promotes osteoporosis is by reducing bone marrow lymphocytes. Since the risk for developing osteoporosis is reportedly greater in smokers with polymorphisms in LRP5, a gene involved in canonical Wnt signaling that regulates bone metabolism, smoking-induced effects on lymphocytes may be influenced by Lrp5 functionality.

RAG and HMGB1 proteins: purification and biochemical analysis of recombination signal complexes.

Two lymphoid cell-specific proteins, called RAG-1 and RAG-2, initiate the process of antigen receptor gene rearrangement, termed V(D)J recombination, by assembling a protein-DNA complex with two recombination signal sequences (RSSs), each of which adjoins a different receptor gene segment, and then introducing a DNA double strand break at the end of each RSS. The study of RAG-RSS complex assembly and activity has been facilitated by the development of methods to purify the RAG proteins and members of the HMG-box family of high mobility group proteins such as HMGB1 that promote RAG binding and cleavage activity in vitro. This chapter describes the purification of recombinant truncated and full-length RAG-1 and RAG-2 expressed transiently in mammalian cells, as well as the purification of bacterially expressed full-length HMGB1.

Identification and characterization of a gain-of-function RAG-1 mutant.

RAG-1 and RAG-2 initiate V(D)J recombination by cleaving DNA at recombination signal sequences through sequential nicking and transesterification reactions to yield blunt signal ends and coding ends terminating in a DNA hairpin structure. Ubiquitous DNA repair factors then mediate the rejoining of broken DNA. V(D)J recombination adheres to the 12/23 rule, which limits rearrangement to signal sequences bearing different lengths of DNA (12 or 23 base pairs) between the conserved heptamer and nonamer sequences to which the RAG proteins bind.