A complementary DNA clone (designated GAT-1) encoding a transporter for the neurotransmitter gamma-aminobutyric acid (GABA) has been isolated from rat brain, and its functional properties have been examined in Xenopus oocytes. Oocytes injected with GAT-1 synthetic messenger RNA accumulated [3H]GABA to levels above control values. The transporter encoded by GAT-1 has a high affinity for GABA, is sodium-and chloride-dependent, and is pharmacologically similar to neuronal GABA transporters. The GAT-1 protein shares antigenic determinants with a native rat brain GABA transporter. The nucleotide sequence of GAT-1 predicts a protein of 599 amino acids with a molecular weight of 67 kilodaltons. Hydropathy analysis of the deduced protein suggests multiple transmembrane regions, a feature shared by several cloned transporters; however, database searches indicate that GAT-1 is not homologous to any previously identified proteins. Therefore, GAT-1 appears to be a member of a previously uncharacterized family of transport molecules.

Elevated levels of the p53 cellular tumor antigen have been previously observed in proliferating and transformed mammalian cells. We found that nontransformed mouse cells treated with either UV light or a UV-mimetic chemical carcinogen exhibited a rapid increase in the amount of p53. This stimulation can be explained, at least in part, on the basis of a post-translational stabilization of p53 which is independent of replicative DNA synthesis, consistent with p53 not being an adventitious product of proliferating cells. The results presented here are interpreted in light of the general hypothesis that p53 is involved in the preparation of mammalian cells for DNA synthesis.The p53 cellular tumor antigen is a cellular protein originally observed (14, 17) in murine cells transformed by the DNA tumor virus simian virus 40 (SV40). Subsequently, it has been demonstrated that the levels of this protein are substantially elevated in a variety of systems (for a review, see reference 11), including cells of different mammalian species which have been neoplastically transformed by DNA tumor viruses, isolated from naturally occurring tumors, or stimulated by either mitogens or serum factors to synthesize DNA. The correlation between expression of high levels of p53 and some transformation events or other treatments stimulating cellular growth is reasonably firm and consistent with the hypothesis that p53 functions in the regulation of the transition of mammalian cells to active proliferation. However, the molecular mechanism by which p53 may affect such a transition is at present unknown.The regulation of expression, as well as the possible function, of p53 in neoplastically transformed cells has been most extensively examined in mouse cells transformed by SV40. It has been established that the elevated levels of p53 observed in SV40-transformed mouse cells are a function of the tight interaction (14,17,21,24) of this protein with the SV40-encoded large tumor antigen (T-Ag); this interaction manifests itself in a dramatic increase in the half-lives of both proteins (24). Because of these facts and other demonstrations that the levels of p53 are increased in DNA tumor virus-transformed and proliferating cells, it has been suggested that pS3 functions in the regulation of cellular growth by interacting with viral transforming gene products (22) in virus-transformed cells and, in normal cells, by interacting with a cellular protein(s) (7,15).In general, it is speculated that the precise molecular function of p53 in affecting the transition of cells to active DNA synthesis has to do with the initiation of DNA replication. This suggestion is supported by several lines of evidence: (i) the viral proteins with which p53 interacts in DNA tumor virus-transformed cells are also required for viral DNA replication (8,10,25,29,30), (ii) the stimuli which increase the levels of p53 in nontransformed cells also induce DNA synthesis (7,22), and (iii) cellular transformation or neoplasia can generally be viewed as involving an abrogation of ...

No abstract

V(D)J recombination is responsible for the de novo creation of antigen receptor genes in T-and B-cell precursors. To the extent that lymphopoiesis takes place throughout an animal's lifetime, recombination errors present an ongoing problem. One type of aberrant rearrangement ensues when DNA sequences resembling a V(D)J joining signal are targeted by mistake. This study investigates the type of sequence likely to be subject to mistargeting, the level of joining-signal function associated with these sequences, and the number of such cryptic joining signals in the genome.In a vertebrate, many millions of B and T cells are produced daily. A pivotal event in B-and T-cell differentiation is the assembly, through DNA recombination, of antigen receptor genes (reviewed in reference 43). Recombination is mediated by two key proteins, called RAG1 and RAG2, as well as an incompletely defined collection of other enzymes and factors (reviewed in references 28 and 56). During pre-B-cell and pre-T-cell development, DNA segments termed V, D, and J are assembled to form the variable exon of the expressed immunoglobulin (Ig) and T-cell receptor (TCR) genes. The joined gene segments encode the highly variable antigen-binding domain of the antigen receptor protein.V(D)J assembly is accomplished through DNA rearrangements that can encompass up to several megabases. The sequence motifs that serve as recognition elements for this process (joining signals) are surprisingly compact. A joining signal, found adjacent to every V, D, or J segment, is comprised of a heptamer (CACAGTG), a spacer (of 12 or 23 bp), and a nonamer (ACAAAAACC) (Fig. 1A). No sequence apart from the 28 (or 39)-bp joining signal motif is required for sitespecific recognition and recombination (reference 33 and references cited therein).Any V(D)J recombination event is normally an interaction between two gene segments, one of which possesses a 12-bp spacer (12-spacer) signal, and the other of which possesses a 23-bp spacer (23-spacer) signal. The molecular signature of V(D)J recombination is production of two types of junctions. One of these, the coding joint, is formed from the fusion of the two coding sequences (e.g., V and J), and the other, a signal joint, is formed from the corresponding joining signals. It is usual for a coding joint to have a small and variable degree of base loss and insertion at the point where the two coding elements connect, whereas the signal joint is typified by a precise fusion, heptamer to heptamer, of the two signals.Not surprisingly, DNA sequences that resemble an authentic joining signal (cryptic signals) are sometimes rearranged by mistake (first described in reference 36). The result is the production of recombinant junctions that are structurally analogous to coding joints and signal joints (Fig. 1) (17, 36). In this report, we take production of the characteristic junctions as the defining, diagnostic feature of a cryptic signal.Cryptic signals are seen at several of the Ig and TCR loci in both mice and humans and are active in re...

Tris+/Na + permeability ratios were measured from shifts in the biionic reversal potentials of the macroscopic ACh-induced currents for 3 wild-type (WT), 1 hybrid, 2 subunit-deficient, and 25 mutant nicotinic receptors expressed in Xenopus oocytes. At two positions near the putative intracellular end of M2, 2' ({xThr244, 13Gly255, -yThr253, 8Ser258) and -1', point mutations reduced the relative Tris + permeability of the mouse receptor as much as threefold. Comparable mutations at several other positions had no effects on relative Tris ÷ permeability. Mutations in ~ had a greater effect on relative Tris + permeability than did comparable mutations in ~/; omission of the mouse ~ subunit (~0 receptor) or replacement of mouse ~ with Xenopus ~ dramatically reduced relative Tris + permeability. The WT mouse muscle receptor (a15~8) had a higher relative permeability to Tris ÷ than the wild-type Torpedo receptor. Analyses of the data show that (a) changes in the Tris+/Na + permeability ratio produced by mutations correlate better with the hydrophobicity of the amino acid residues in M2 than with their volume; and (b) the mole-fraction dependence of the reversal potential in mixed Na+/Tris + solutions is approximately consistent with the Goldman-Hodgkin-Katz voltage equation. The results suggest that the main ion selectivity filter for large monovalent cations in the ACh receptor channel is the region delimited by positions -1' and 2' near the intracellular end of the M2 helix.