A novel fluorescence-based system for assaying and separating live cells according to VDJ recombinase activity.

Yancopouloš, George D.; Nolan, Garry P.; Pollock, Roberta R.; Prockop, Susan E.; Li, S C; Herzenberg, L A; Alt, Frederick W.

doi:10.1128/mcb.10.4.1697

Cited by 24 publications

(16 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In this process, specific signal sequences that flank each of the possible coding regions act as targets for the recombination event. The recombination activity is limited to cell lines representing precursors of B and T lymphocytes (2,3) and is absent in mature cells that produce immunoglobulin or T-cell receptor molecules. The proteins involved in V(D)J recombination have not been characterized biochemically, but recently two lymphoidspecific genes have been identified as playing a crucial role in the process.…”

Section: Abstract V(d)j [Variable-(diversity)-joining] Recom-mentioning

confidence: 99%

V(D)J recombination activity in lymphoid cell lines is increased by agents that elevate cAMP.

Menetski

Gellert

1990

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

V(D)J [variable-(diversity)-joining] recom-bination is regulated developmentally, being restricted to cells of the early B-and T-lymphocyte lineages. In this report we show that recombination activity can also be regulated in response to chemical effectors. Compounds that increase intracellular cAMP increase V(D)J recombination of extrachromosomal substrates in pre-B-cell lines as much as 10-fold. In contrast, V(D)J recombination is reduced 5-to 8-fold in response to phorbol 12-myristate 13-acetate or to the calcium ionophore A23187. The effect of cAMP agonists on recombination appears to reflect an increase in cellular recombination activity, as indicated by the caffeine-induced rise in the level of mRNA from the recombination-activating genes RAG) and RAG2. Our data demonstrate that intracellular second messengers modulate recombination activity in lymphoid cell lines, implying that recombination activity can be regulated by these signals in developing B and T lymphocytes.The assembly of functional immunoglobulin and T-cell receptor genes is carried out by a series of site-specific recombination events known as V(D)J [variable-(diversity)-joining] recombination (1). In this process, specific signal sequences that flank each of the possible coding regions act as targets for the recombination event. The recombination activity is limited to cell lines representing precursors of B and T lymphocytes (2,3) and is absent in mature cells that produce immunoglobulin or T-cell receptor molecules. The proteins involved in V(D)J recombination have not been characterized biochemically, but recently two lymphoidspecific genes have been identified as playing a crucial role in the process. The products of the RAGI and RAG2 genes, when expressed in fibroblasts, can activate V(D)J recombination in those cells and may well be directly involved in the reaction (4, 5).Lymphoid cells have been shown to be responsive to many different extracellular factors such as lipopolysaccharides, lymphokines, neurotransmitters, histamine, and growth factors (6). Various cell lines respond to these factors by making new surface markers (7), producing lymphokines (8), transcribing specific genes (9), and activating DNA-binding proteins (10). In several cases, the intracellular second messengers of extracellular factors have been determined. Abraham et al. (11) have shown that interleukin 2 production in a T-cell line is induced by interleukin 1 and phytohemagglutinin, and this induction can be mimicked by factors that stimulate protein kinase C (PKC) and increase intracellular calcium. Similarly, K light-chain enhancer binding activity and lightchain transcription can be induced by interleukin 1 or agents that increase intracellular cAMP concentration in certain B-cell lines (10). Thus, many enzyme systems in lymphocytes are influenced by changes in various second-messenger pathways.In this study we have tested agents that affect secondmessenger pathways for their influence on V(D)J recombination activity of several lymphoid cell lines. ...

show abstract

Section: Abstract V(d)j [Variable-(diversity)-joining] Recom-mentioning

confidence: 99%

V(D)J recombination activity in lymphoid cell lines is increased by agents that elevate cAMP.

Menetski

Gellert

1990

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…FACS analysis has been used to enrich cells stably transfected with the P-Gal gene driven by various promoters (24)(25)(26)(27)(28). We adapted this procedure for use with cells transiently transfected with a ,3-Gal expression vector (pCH110), as well as pLTRluc, containing the full-length MMTV LTR-driving luciferase, and the PR expression vector, as shown in Fig.…”

mentioning

confidence: 99%

Newly expressed progesterone receptor cannot activate stable, replicated mouse mammary tumor virus templates but acquires transactivation potential upon continuous expression.

Smith

Archer

Hamlin-Green

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

During development and differentiation, the expression of banscription factors is regulated in a temporal fashion. Newly expressed transcription factors must interact productively with target genes organized in chromatin. Although the mechanisms governing factor binding to chromatin templates are not well understood, it is now clear that template access can be dramatically influenced by nudeoprotein structure. We have examined the ability of a well characterized ranwactivator, the progesterone receptor (PR), to activate the mouse mammna tumor virus (MMTV) promoter organized either in stable, replicating templates that have a highly ordered nucleosome structure or as transiently transfected DNA, which adopts a less-dermed structure. (8). In this structural configuration, nuclear factor 1 (NF1) is excluded from its target site in the proximal promoter (1, 6, 9, 10). Binding of the activated glucocorticoid receptor (GR) to the promoter induces a chromatin remodeling event associated with the second nucleosome (Nuc-B) (8, 11). In addition, histone Hi is depleted from the promoter proximal region in a hormone-dependent manner (12). We proposed previously that this chromatin transition is directly and mechanistically involved in the binding of NF1 and subsequent formation of the transcription preinitiation complex (9, 10). Similar phenomena have been observed for the murine tyrosine aminotransferase (13-15) and yeast phoS (2,16) genes.The progesterone receptor (PR) and GR activate the MMTV promoter through the same target sequences as determined by transient transfections (17)(18)(19)(20)

show abstract

“…Analyses of transfected recombination substrates have demonstrated that this activity is restricted to pre-B-and pre-T-cell lines; activity has not been detected in nonlymphoid cells or in cells that represent more mature stages of the lymphocyte lineages (13,30). Two genes that synergistically confer V(D)J recombination activity to nonlymphoid cells have been isolated and referred to as recombination-activating genes 1 and 2 (RAG-1 and RAG-2, respectively) (18,23).…”

mentioning

confidence: 99%

A V(D)J recombinase-inducible B-cell line: role of transcriptional enhancer elements in directing V(D)J recombination.

et al. 1993

Self Cite

View full text Add to dashboard Cite

Rapid analysis of mechanisms that regulate V(D)J recombination has been hampered by the lack of appropriate cell systems that reproduce aspects of normal prelymphocyte physiology in which the recombinase is activated, accessible antigen receptor loci are rearranged, and rearrangement status is fixed by termination of recombinase expression. To generate such a system, we introduced heat shock-inducible V(D)J recombination-activating genes (RAG) 1 and 2 into a recombinationally inert B-cell line. Heat shock treatment of these cells rapidly induced high levels of RAG transcripts and RAG proteins that were accompanied by a parallel induction of V(D)J recombinase activity, strongly suggesting that RAG proteins have a primary role in V(D)J recombination. Within hours after induction, these cells began to rearrange chromosomally integrated V(D)J recombination substrates but only if the substrates contained an active transcriptional enhancer, substrates lacking an enhancer were not efficiently rearranged. Activities necessary to target integrated substrates for rearrangement were provided by two separate lymphoid-specific transcriptional enhancers, as well as an active nonlymphoid enhancer, unequivocally demonstrating that such elements enhance both transcription and V(D)J recombinational accessibility.Precursor lymphocytes (prelymphocytes) assemble antigen receptor variable region genes from an array of variable (V), diversity (D), and joining (J) gene segments via a site-specific enzymatic activity termed V(D)J recombinase (reviewed in reference 4). Analyses of transfected recombination substrates have demonstrated that this activity is restricted to pre-B-and pre-T-cell lines; activity has not been detected in nonlymphoid cells or in cells that represent more mature stages of the lymphocyte lineages (13,30). Two genes that synergistically confer V(D)J recombination activity to nonlymphoid cells have been isolated and referred to as recombination-activating genes 1 and 2 (RAG-1 and RAG-2, respectively) (18, 23). The precise function of the RAG products remains unknown; they may encode tissuespecific components of the V(D)J recombinase system or may serve to induce or activate components of the V(D)J recombination complex. However, gene targeting experiments have provided conclusive evidence that coexpression of the RAG-1 and RAG-2 products in developing lymphocytes is essential to generate V(D)J recombinase activity (17,26).Assembly of V gene segments is a highly ordered process and is controlled at several levels, including cell lineage specificity, the stage of lymphocyte differentiation, and allelic exclusion (reviewed in reference 2). Because all V(D)J rearrangement events are effected by a common recombinase which is present throughout prelymphocyte development (2, 29), targeting of this activity must be controlled by modulating the accessibility of substrate gene segments (5). In turn, accessibility has been correlated with active transcription of V gene segments, but the precise role of tran-* Corresponding aut...

show abstract

A novel fluorescence-based system for assaying and separating live cells according to VDJ recombinase activity.

Cited by 24 publications

References 22 publications

V(D)J recombination activity in lymphoid cell lines is increased by agents that elevate cAMP.

V(D)J recombination activity in lymphoid cell lines is increased by agents that elevate cAMP.

Newly expressed progesterone receptor cannot activate stable, replicated mouse mammary tumor virus templates but acquires transactivation potential upon continuous expression.

A V(D)J recombinase-inducible B-cell line: role of transcriptional enhancer elements in directing V(D)J recombination.

Contact Info

Product

Resources

About