of Microbiology and Immunology University of California Berkeley, California 94720 Summary DNA-nuclear protein interactions were studied with synthetic recombination signal sequences (RSSs) for immunoglobulin V-J joining. With a gel retardation as- say, a DNA-binding protein that specifically interacts with RSSs was detected in nuclear extracts from a pre- B cell line, 3889. This protein was found in all the recombination-competent pre-B cell lines tested in this study, but not in myeloma, mature T cell, mono- cyte, or fibroblast cell lines. DNA footprint analysis with dimethyl sulfate demonstrated that the 7-mer re- gion of the RSS was strongly protected when com- plexed with the binding protein. Furthermore, a single base substitution in the Fmer region totally abolished the binding. The molecular mechanism of V-J joining is discussed in the context of the RSS-binding protein. Introduction Antigen receptor genes are generated by site-specific DNA recombination both in T cells and 6 cells during the differentiation of lymphocytes (reviewed by Kronenberg et al., 1986; Tonegawa, 1983). On the germ-line chromo- some, variable region genes for the antigen receptor are split into variable (V), diversity (D), and joining (J) seg- ments. These gene segments are brought together by a site-specific DNA recombination process known as V-(D)- J joining. This DNA rearrangement plays a key role not only in the activation of the antigen receptor genes, but also in the diversification of the gene sequences. To study the molecular mechanism of V-(D)-J joining, three different approaches have been taken. One ap- proach is the structural study of the recombination region by DNA cloning and sequencing. From these analyses it has been revealed that both immunoglobulin (lg) and T cell receptor (TCR) genes contain two highly conserved sequences adjacent to the recombination sites; they are a palindromic 7-mer, CACTGTG, and a T-rich 9-mer, GGTTTTTGT, separated by a spacer of constant length (Sakano et al., 1979, 1980, 1981; Max et al., 1979; Early et al., 1980; Davis, 1985). The second approach is to in- troduce artificial recombination substrates with appropri- ate selection markers into recombination-competent pre-B cells (Lewis et al., 1984, 1985; Blackwell and Alt, 1984; Yancopoulos et al., 1986; Hesse et al., 1987). Alt and Balti- more and their colleagues have demonstrated that V-(D)-J joining can take place on exogenous lg or TCR genes in- troduced into the host chromosomal DNA with a retroviral vector or by DNA transfection. The third approach is to characterize the enzymatic machinery responsible for V-(D)-J joining. We assume that at least three activities are involved in the recombinase complex for V-(D)-J joining (Hope et al., 1986): a DNA-binding activity, an endonu- cleolytic activity, and a ligase activity. The DNA-binding activity would recognize the recombination sites and bring the two substrates together into the proper orientation; the endonucleolytic activity would cleave the germ-line seg- ments at the recombination sites; and finally, the ligase activity would join the two cleaved DNA substrates cova- lently to complete the joining reaction. Endonucleases presumably involved in the DNA-cutting activity of recombinase have been previously described (Desiderio and Baltimore, 1984; Kataoka et al., Hope 1986). However, little is known about other activities, and as yet the identification of a whole recombinase com- plex has not been reported. In the present study we have attempted to identify a DNA-binding protein(s) that interacts specifically with recombination signal sequences (RSSs). As a source of the protein, we have used nuclear extracts from an immature pre-B cell line, 3889, kindly provided by Dr. F. W. Alt. In this cell line lg gene rearrangement actively takes place during in vitro culture (Alt et al., 1981; Black- well and Alt, 1984). We synthesized DNA substrates con- taining the Fmer and 9-mer separated by either a 12 bp or a 23 bp spacer. Using the synthetic recombination sub- strates, we first verified that two sets of RSSs are sufficient to cause the V-J-type joining provided the 12/23 bp spacer rule (Sakano et al., 1980; Early et al., 1980) is satisfied. We then searched for a specific DNA-binding protein(s) in the pre-B cell nuclear extracts by using the synthesized RSS probes. With a gel retardation assay (Singh et al., 1986; Schneider et al., 1986), we have identified a nuclear pro- tein that can specifically interact, particularly in the Fmer region, with the RSS probe containing the 12 bp spacer. In this report we characterize this RSS-binding activity, which may be one of the DNA-binding components as- sociated with V-(D)-J joining. Results Synthetic Substrates Containing the Fmer and 9-mer Can Cause V-J-Type Recombination Two double-stranded oligonucleotides containing the Fmer and 9-mer were chemically synthesized (Figure 1A). One contained a 12 bp spacer separating the recombina- tion signals (12 bp-RSS), and the other contained a 23 bp spacer (23 bp-RSS). To study whether these two RSSs are sufficient substrates to mediate V-J-type recombina- tion, we constructed a plasmid, pKOdel-1. It contains a ne- omycin resistance gene (near) (Beck et al., 1982) flanked by two sets of RSSs, and a thymidine kinase gene (tk) (McKnight, 1980) further downstream (Figure 1B). Expres- sion of these genes is mediated by a promotor complex consisting of the SV40 and t/r gene promoters (McKnight, 1980; Southern and Berg, 1982).
Toll-like receptors (TLRs) sense invading microbial pathogens and play crucial roles in the activation of innate and adaptive immunity. However, excessive TLR activation can disrupt immune homeostasis, and may be responsible for the development of autoimmune and inflammatory diseases. As such, the molecules and pathways that negatively control TLR signaling have been intensively investigated. Here, we discuss recent insights into the negative regulation of TLR signaling, with focus on three major mechanisms: (i) dissociation of adaptor complexes; (ii) degradation of signal proteins; and (iii) transcriptional regulation. We also highlight how pathogens negatively target TLR signaling as a strategy to evade the host immune response.
Toll-like receptors (TLRs) play a critical role in the detection of invading pathogens within the body and the subsequent immune response. Individual TLRs recognize distinct microbial components. The TLRs are a type 1 transmembrane receptor that possess an extracellular leucine-rich repeat domain and cytoplasmic domain homologous with that of the interleukin 1 receptor (IL-1R) family. Upon stimulation, TLR recruits the IL-1R-associated kinase (IRAK) via the adapter MyD88, ultimately leading to the activation of nuclear factor-κB. Cytokine production in response to all TLR ligands is completely abolished in MyD88-deficient cells, indicating that MyD88 is an essential signalling molecule shared among members of the IL-1R/Toll family. However, several novel adaptor molecules have recently been identified. Evidence is now accumulating showing that differential utilization of these adaptors may activate overlapping as well as distinct signalling pathways, and ultimately give rise to distinct biological effects exerted by individual TLR family members.
