Biomedical subjects

F Weis-Garcia

Publications and source records attributed to F Weis-Garcia.

5 recordsLinked to original sources

PubMed1997

V(D)J recombination: modulation of RAG1 and RAG2 cleavage activity on 12/23 substrates by whole cell extract and DNA-bending proteins.

Antigen receptor gene rearrangement is directed by DNA motifs consisting of a conserved heptamer and nonamer separated by a nonconserved spacer of either 12 or 23 base pairs (12 or 23 recombination signal sequences [RSS]). V(D)J recombination requires that the rearranging DNA segments be flanked by RSSs of different spacer lengths, a phenomenon known as the 12/23 rule. Recent studies have shown that this restriction operates at the level of DNA cleavage, which is mediated by the products of the recombination activating genes RAG1 and RAG2. Here, we show that RAG1 and RAG2 are not sufficient for 12/23 dependent cleavage, whereas RAG1 and RAG2 complemented with whole cell extract faithfully recapitulates the 12/23 rule. In addition, HMG box containing proteins HMG1 and HMG2 enhance RAG1- and RAG2-mediated cleavage of substrates containing 23 RSS but not of substrates containing only 12 RSS. These results suggest the existence of a nucleoprotein complex at the cleavage site, consisting of architectural, catalytic, and regulatory components.

DNA↗

PubMed1997

V(D)J recombination: in vitro coding joint formation.

Antigen receptor genes are assembled through a mechanism known as V(D)J recombination, which involves two different joining reactions: signal and coding joining. Formation of these joints is essential for antigen receptor assembly as well as maintaining chromosomal integrity. Here we report on a cell-free system for coding joint formation using deletion and inversion recombination substrates. In vitro coding joint formation requires RAG1, RAG2, and heat-labile factors present in the nuclear extract of nonlymphoid cells. Both inversion- and deletion-mediated coding joint reactions produce diverse coding joints, with deletions and P nucleotide addition. We also show that deletion-mediated coding joint formation follows the 12/23 rule and requires the catalytic subunit of DNA-dependent protein kinase.

Cell Nucleus↗

PubMed1996

In vitro V(D)J recombination: signal joint formation.

The first step of V(D)J recombination, specific cleavage at the recombination signal sequence (RSS), can be carried out by the recombination activating proteins RAG1 and RAG2. In vivo, the cleaved coding and signal ends must be rejoined to generate functional antigen receptors and maintain chromosomal integrity. We have investigated signal joint formation using deletion and inversion substrates in a cell free system. RAG1 and RAG2 alone or in combination were unable to generate signal joints. However, RAG1 and RAG2 complemented with nuclear extracts were able to recombine an extrachromosomal substrate and form precise signal joints. The in vitro reaction resembled authentic V(D)J recombination in being Ku-antigen-dependent.

Animals↗

PubMed1996

Complementation between kinase-defective and activation-defective TGF-beta receptors reveals a novel form of receptor cooperativity essential for signaling.

Transforming growth factor-beta (TGF-beta) signals through two transmembrane serine/threonine kinases, T beta R-I and T beta R-II. TGF-beta binds to T beta R-II, allowing this receptor to associate with and phosphorylate T beta R-I which then propagates the signal. T beta R-I is phosphorylated within its GS domain, a region immediately preceding the kinase domain. To further understand the function of T beta R-I in this complex, we analyzed T beta R-I-inactivating mutations identified in cell lines that are defective in TGF-beta signaling yet retain ligand binding ability. The three mutations identified here all fall in the kinase domain of T beta R-I. One mutation disrupts the kinase activity of T beta R-I, whereas the other two mutations prevent ligand-induced T beta R-I phosphorylation, and thus activation, by T beta R-II. Unexpectedly, a kinase-defective T beta R-I mutant can functionally complement an activation- defective T beta R-I mutant, by rescuing its T beta R-II- dependent phosphorylation. Together with evidence that the ligand-induced receptor complex contains two or more T beta R-I molecules, these results support a model in which the kinase domain of one T beta R-I molecule interacts with the GS domain of another, enabling its phosphorylation and activation by T beta R-II. This cooperative interaction between T beta R-I molecules appears essential for TGF-beta signal transduction.

Amino Acid Sequence↗

PubMed1996

Serine/threonine kinase receptors: mediators of transforming growth factor beta family signals.

TGF beta represents the largest and most versatile cytokine family known in metazoans. The recent identification of transmembrane serine/threonine kinases as TGF beta family receptors represents a major milestone towards understanding how these factors elicit their varied responses. Genetic and biochemical evidence suggests a general model for the mechanism of activation of these receptors. In this model, the ligand acts as an adaptor, bringing a primary receptor kinase in contact with a second kinase, which becomes phosphorylated and thereby competent to propagate the signal to downstream components. Such a kinase cascade on the membrane defines a new variation in signal transduction. Although many details of this mechanism remain to be clarified, its combinatorial capacity may explain the multifunctional nature of its ligands. Several key genes have been identified whose regulation by these signals mediates cellular responses such as cell cycle arrest. This field is advancing at a fast pace, and the identity of components that carry the receptor signals to these genes should soon be unveiled. It is expected that these advances, as they unfold, will in turn clarify the role of miscreant TGF beta signalling in human diseases, cancer included.

Animals↗