Self-sparing of long-term in vitro-cloned or uncloned cytotoxic T lymphocytes.

At least some long-term in vitro-cultured cytotoxic T cell clones and uncloned cell populations are able, in the presence of Con A, to lyse other cells, to be lysed by other cells, but not to lyse themselves. This as-yet-unexplained result may have implications as to the mechanism of T cell-mediated cytotoxicity.

A novel calmodulin-binding protein, belonging to the WD-repeat family, is localized in dendrites of a subset of CNS neurons.

A rat brain synaptosomal protein of 110,000 M(r) present in a fraction highly enriched in adenylyl cyclase activity was microsequenced (Castets, F., G. Baillat, S. Mirzoeva, K. Mabrouk, J. Garin, J. d'Alayer, and A. Monneron. 1994. Biochemistry. 33:5063-5069). Peptide sequences were used to clone a cDNA encoding a novel, 780-amino acid protein named striatin. Striatin is a member of the WD-repeat family (Neer, E.J., C.J. Schmidt, R. Nambudripad, and T.F. Smith. 1994. Nature (Lond.). 371:297-300), the first one known to bind calmodulin (CaM) in the presence of Ca++. Subcellular fractionation shows that striatin is a membrane-associated, Lubrol-soluble protein. As analyzed by Northern blots, in situ hybridization, and immunocytochemistry, striatin is localized in the central nervous system, where it is confined to a subset of neurons, many of which are associated with the motor system. In particular, striatin is conspicuous in the dorsal part of the striatum, as well as in motoneurons. Furthermore, striatin is essentially found in dendrites, but not in axons, and is most abundant in dendritic spines. We propose that striatin interacts, through its WD-repeat domain and in a CaM/Ca(++)-dependent manner, with one or several members of a surrounding cluster of molecules engaged in a Ca(++)-signaling pathway specific to excitatory synapses.

Calcium signalling in Bacillus subtilis.

Few systematic studies have been devoted to investigating the role of Ca2+ as an intracellular messenger in prokaryotes. Here we report an investigation on the potential involvement of Ca2+ in signalling in Bacillus subtilis, a Gram-positive bacterium. Using aequorin, it is shown that B. subtilis cells tightly regulate intracellular Ca2+ levels. This homeostasis can be changed by an external stimulus such as hydrogen peroxide, pointing to a relationship between oxidative stress and Ca2+ signalling. Also, B. subtilis growth appears to be intimately linked to the presence of Ca2+, as normal growth can be immediately restored by adding Ca2+ to an almost non-growing culture in EGTA containing Luria broth medium. Addition of Fe2+ or Mn2+ also restores growth, but with 5-6 h delay, whereas Mg2+ did not have any effect. In addition, the expression of alkyl hydroperoxide reductase C (AhpC), which is strongly enhanced in bacteria grown in the presence of EGTA, also appears to be regulated by Ca2+. Finally, using 45Ca2+ overlay on membrane electrotransferred two-dimensional gels of B. subtilis, four putative Ca2+ binding proteins were found, including AhpC. Our results provide strong evidence for a regulatory role for Ca2+ in bacterial cells.

Inventory, assembly and analysis of Bacillus subtilis ABC transport systems.

We have undertaken the inventory and assembly of the ATP binding cassette (ABC) transporter systems in the complete genome of Bacillus subtilis. We combined the identification of the three protein partners that compose an ABC transporter (nucleotide-binding domain, NBD; membrane spanning domain, MSD; and solute-binding protein, SBP) with constraints on the genetic organization. This strategy allowed the identification of 86 NBDs in 78 proteins, 103 MSD proteins and 37 SBPs. The analysis of transcriptional units allows the reconstruction of 59 ABC transporters, which include at least one NBD and one MSD. A particular class of five dimeric ATPases was not associated to MSD partners and is assumed to be involved either in macrolide resistance or regulation of translation elongation. In addition, we have detected five genes encoding ATPases without any gene coding for MSD protein in their neighborhood and 11 operons that encode only the membrane and solute-binding proteins. On the bases of similarities, three ATP-binding proteins are proposed to energize ten incomplete systems, suggesting that one ATPase may be recruited by more than one transporter. Finally, we estimate that the B. subtilis genome encodes for at least 78 ABC transporters that have been split in 38 importers and 40 extruders. The ABC systems have been further classified into 11 sub-families according to the tree obtained from the NBDs and the clustering of the MSDs and the SBPs. Comparisons with Escherichia coli show that the extruders are over-represented in B. subtilis, corresponding to an expansion of the sub-families of antibiotic and drug resistance systems.

An automatic approach for DNA sequencing.

Automation is perfectly suited to DNA sequencing, which requires many repetitive steps and the handling of many samples. It increases not only processing capability but also accuracy and reproducibility. We present an approach to automation of DNA sequencing that was developed at the level of a research laboratory, and describe several improvements to DNA sequencing methodology.

Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability.

A convenient way to estimate the number of viable cells growing in microtitre tray wells is to use a colorimetric assay and an automatic microplate scanning spectrophotometer. One such assay, developed by Mosmann, depends on the reduction by living cells of tetrazolium salt, MTT, to form a blue formazan product. However the original technique has several technical limitations, namely a less than optimal sensitivity, a variable background due to protein precipitation on adding an organic solvent to dissolve the blue formazan product, and a low solubility of the product. These problems have been overcome by the following modifications: avoidance of serum in the incubation medium, thus overcoming precipitation problems in the organic solvent; avoidance of phenol red in the incubation medium, thus avoiding the use of acid in the final solvent which altered the spectral properties of the formazan; elimination of the medium containing MTT after the reaction and subsequent use of pure propanol or ethanol to rapidly solubilize the formazan; use of a higher concentration of MTT; use of half-area microtitre trays to increase the spectrophotometer readings from a given amount of formazan; use of a more judicious reference wavelength in a dual wavelength spectrophotometer. With these modifications the reliability and sensitivity of the test have been increased to the point where it can in many cases replace the [3H]thymidine uptake assay to measure cell proliferation or survival in growth factor or cytotoxicity assays. Examples of its use in IL-2 assays are given.

Xenoserum-induced cytolytic "T" cells: polyclonal specificity with an apparent "anti-self" component, and cooperative induction.

Mice were primed in vivo by injection of fetal calf serum (FCS) and their spleen cells were incubated in vitro for 5 days in medium containing 10% FCS. This resulted in the development of cytolytic activity, which was most probably due to "T" cells, since effector cells 1) were sensitive to anti-Thy 1 antiserum or monoclonal antibodies in the presence of complement, 2) were not retained on Ig-anti Ig columns, 3) did not develop from "nude" spleen cells. Further arguments for the T cell nature of these effector cells came from their specificity. Blocking experiments using unlabeled competitor cells demonstrated that FCS-induced cytolysis was polyclonal, with clones recognizing allogeneic or syngeneic determinants possibly related to allo or self H-2. In keeping with polyclonality, cytolysis tested on any given target cell was greatly increased by adding Concanavalin A during the cytolysis test. Experiments were made to investigate whether in particular the anti-self cytolytic activity was directed against FCS determinants. We feel that this possibility, although not formally excluded, was made unlikely. The polyclonal specificity at the effector stage stood in sharp contrast to the serum specificity at the induction stage (reported elsewhere). We demonstrated that these two sets of specificities corresponded to two sets of specific cells. A first population of FCS-primed cells had "promoter" activity, in the sense that it could trigger a second population of "precursor" cells to differentiate into polyclonally cytolytic T cells.

Rapid orientated cloning in a shuttle vector allowing modulated gene expression in Bacillus subtilis.

An expression vector for systematic protein overproduction in Bacillus subtilis has been constructed. It derives from pDG148 and combines the main property of this vector, i.e. conditional expression of the gene in response to isopropylbeta-D-thiogalactopyranoside, with (i) rapid orientated cloning by a ligation independent procedure and (ii) a ribosome binding site of high translational efficiency. When used for overproduction of several proteins in B. subtilis, this vector gave good levels of protein synthesis.

Primary in vitro mouse B-cell response induced by self-hormone-coupled self-albumin.

[3H'-thymidine incorporation above control levels was observed when normal mouse spleen cells were cultured with pituitary hormone-coupled albumins. Most striking was the observation of proliferation with self-hormone-coupled self-albumin, for instance vasopressin-coupled mouse serum albumin. The proliferating cells were not sensitive to anti-Thy 1 antiserum plus complement and were present in nude mouse spleens. Proliferation was accompanied with the appearance of antibody-forming cells against an irrelevant antigen (sheep red blood cells). These results strongly suggest polyclonal induction of B-cell proliferation/differentiation by self-hormone-coupled self-albumin.

Murine and human T cell factors that induce the differentiation of normal mouse lymphocytes into cytotoxic cells copurify with interleukin 2.

Fetal calf serum (FCS)-specific T promoter cell lines (line 12), clones, or lymphomas produce lymphocyte promoter factors (LPF). These factors are defined as T-cell supernatant activities that induce polyclonal differentiation of normal experimentally unprimed mouse lymphocytes into antibody-forming cells (B-LPF) or into cytotoxic cells (T-LPF). The cytotoxic cells thus induced lysed a broad range of target cells including syngeneic and allogeneic tumour cells and lymphoblasts. We have investigated whether T cell tumours (mouse or human) other than FCS-specific T promoter cell lines (line 12), clones, or lymphomas produce T-LPF activity, and whether T-LPF activity is related to interleukin 2 (IL-2) activity. We found that the EL4 thymoma cells were high producers of T-LPF and IL-2 activity. When EL4 cells and T-LPF+ line 12 lymphomas were cloned, all T-LPF high-producer clones were also high IL-2 producers. In addition, the human Jurkat T tumour cells produced both T-LPF and IL-2 activity which could be detected on both mouse and human lymphocytes. By using biochemical fractionation (size fractionation or chromatofocusing fractionation) and absorption techniques, we could not separate T-LPF and IL-2 activity. Thus, the present data may indicate that the T-LPF and IL-2 activities studied in the present systems are borne by the same molecule(s) (= IL-2?). These results are discussed in relation to current hypotheses on the cellular and molecular requirements for the generation of cytotoxic T cells.

Statistical theory of chromatography: new outlooks for affinity chromatography.

We have developed further the statistical approach to chromatography initiated by Giddings and Eyring, and applied it to affinity chromatography. By means of a convenient expression of moments the convergence towards the Laplace-Gauss distribution has been established. The Gaussian character is not preserved if other causes of dispersion are taken into account, but expressions of moments can be obtained in a generalized form. A simple procedure is deduced for expressing the fundamental constants of the model in terms of purely experimental quantities. Thus, affinity chromatography can be used to determine rate constants of association and dissociation in a range considered as the domain of the stopped-flow methods.

A differential molecular biology search for genes preferentially expressed in functional T lymphocytes: the CTLA genes.

One approach to the isolation of molecules involved in T cell-mediated cytolysis stems from the postulate of a possible correlation between molecular phenotype and molecular functional involvement. Accordingly, CTL-specific molecules have been looked for, using a strategy based on the differential screening of a subtracted cDNA library. This led to the isolation and characterization of the following structures, expressed mostly (but no exclusively) in CTLs and inducible upon lymphocyte activation: CTLA-1 and CTLA-3 (serine-proteases), CTLA-4 (a member of the Ig superfamily) and CTLA-2 alpha and beta (homologues to the proregion of cysteine-proteases). The theoretical and practical limitations and the prospects of this type of approach are discussed.

Isolation and characterization of the lacA gene encoding beta-galactosidase in Bacillus subtilis and a regulator gene, lacR.

We have isolated transposon insertions in the lacA gene encoding an endogenous beta-galactosidase of Bacillus subtilis. Upstream of the putative operon containing lacA is a negative regulator, lacR, which encodes a product related to a family of regulators that includes the lactose repressor, lacI, of Escherichia coli. New strains with insertions in the lacA gene should be of use in studies using lacZ fusions in B. subtilis.

Analysis of errors in finished DNA sequences: the surfactin operon of Bacillus subtilis as an example.

Increased productivity in DNA sequencing would not be valid without a straightforward detection and estimation of errors in finished sequences. The sequence of the surfactin operon from Bacillus subtilis was obtained by two different groups and by chance we were also working on the same chromosome region. Taking advantage of this situation we report in this paper, the number and nature of errors found in the overlapping part of the DNA sequences obtained by the three laboratories. The coincidence of some of the errors with compression in sequence ladders and with secondary DNA structures as well as the detection of frameshift errors using computer programs, are demonstrated. Finally we discuss the definition of a new sequencing strategy that might minimize both the error rate and the cost of sequencing.

Cloning of two novel ABC transporters mapping on human chromosome 9.

The family of ATP binding cassette (ABC) transporters or traffic ATPases is composed of several membrane-associated proteins that transport a great variety of solutes across cellular membranes. Two novel mammalian members of the family, ABC1 and ABC2, have been identified by a PCR-based approach. They belong to a group of traffic ATPases encoded as a single multifunctional protein, such as CFTR, STE 6, and P-glycoproteins. Their peculiar structural features and close relationship to ABC transporters involved in nodulation suggest that ABC1 and ABC2 define a novel subgroup of mammalian traffic ATPases.

Clonal expansion of T cells: a cytotoxic T-cell response in vivo that involves precursor cell proliferation.

The response of peritoneal exudate lymphocytes to allogeneic tumor cells was used to determine whether the in vivo generation of cytotoxic T cells (CTL) involved the proliferation of precursor cells. Ten days post-injection, both cytotoxic activity and the formation of conjugates between lymphocytes and target cells were shown to be specific for the immunizing tumor alloantigens and to be effected by Ly-2+ cells. A cell-sorting-based procedure was developed to isolate specific conjugates between red-fluorescence-tagged CTL and blue-fluorescence-tagged tumor target cells. When [3H]thymidine was administered during the response, almost all isolated conjugate-forming CTL were 3H-labeled on autoradiography. Thus, the CTL were clearly products of dividing cells, a result that contradicts published data. Reassessment of a previously studied system, which suggested that CTL were not products of cell division, indicated that in that system many of the conjugate-forming cytotoxic cells studied were Ly-2- and nonspecific, and thus perhaps not T cells. We conclude that the clonal selection model is applicable to at least one in vivo T-cell response.

CTLA-8, cloned from an activated T cell, bearing AU-rich messenger RNA instability sequences, and homologous to a herpesvirus saimiri gene.

To detect novel molecules involved in immune functions, a subtracted cDNA library between closely related murine lymphoid cells was prepared using improved technology. Differential screening of this library yielded several clones with a very restricted tissue specificity, including one that we named CTLA-8. CTLA-8 transcripts could be detected only in T cell hybridoma clones related to the one used to prepare the library. Southern blots showed that the CTLA-8 gene was single copy in mice, rats, and humans. By radioactive in situ hybridization, the CTLA-8 gene was mapped at a single site on mouse chromosome 1A and human chromosome 2q31, in a known interspecific syntenic region. The CTLA-8 cDNA sequence indicated the presence, in the 3'-untranslated region of the mRNA, of AU-rich repeats previously found in the mRNA of various cytokines, growth factors, and oncogenes. The CTLA-8 cDNA contained an open reading frame encoding a putative protein of 150 amino acids. This protein was 57% homologous to the putative protein encoded by the ORF13 gene of herpesvirus Saimiri, a T lymphotropic virus. These findings are discussed in the context of other genes of this herpesvirus homologous to known immunologically active molecules. More generally, CTLA-8 may belong to the growing set of virus-captured functionally important cellular genes related to the immune system or to cell death and cell survival.

YAC-assisted cloning of a putative G-protein mapping to the MHC class I region.

We report the successful use of whole yeast artificial chromosomes (YACs) as probes for direct positional cloning of novel expressed sequences in a given genomic fragment. The class I region of the human major histocompatibility complex, in particular the chromosomal fragment spanning the HLA-E locus, was investigated. The screening of a cDNA library with a 210-kb-long YAC clone led to the identification of a new gene, positionally conserved in the major histocompatibility complex of the mouse genome and encoding a putative GTP binding protein. Although its precise function remains unknown, the interspecies conservation of both sequence and map position suggests a regulatory or functional link with the histocompatibility cluster.

P815 mastocytoma cells, classical targets for cytotoxic T lymphocytes, exert natural cytotoxicity.

While investigating the tissue distribution of cytotoxic-T-lymphocyte-associated (CTLA) gene transcripts, we found that some of these could be detected in mast cells. This led us to test the cytolysis exerted by a number of mast cell populations. We briefly report here that P815 cells, classically known as excellent target cells for cytotoxic T cells, exert natural cytotoxicity toward WEHI-164 target cells.