Transfected F3/F11 neuronal cell surface protein mediates intercellular adhesion and promotes neurite outgrowth.

The mouse neuronal F3 glycoprotein and its chicken homolog F11 belong to a subclass of proteins of the immunoglobulin superfamily with preferential localization on axons and neurites. We have transfected F3 cDNA into CHO cells. Biochemical analysis establishes that the cDNA we have cloned codes for a 130 kd phosphatidylinositol-anchored polypeptide. F3-expressing transfectants exhibited enhanced self-adhesive properties, aggregating with faster kinetics and forming larger aggregates than F3-negative control cells. When used as a culture substrate for sensory neurons, F3-transfected cells showed a markedly enhanced ability to promote neurite outgrowth compared with nontransfected cells. The results support the idea that F3/F11 and other closely similar proteins function as cell adhesion molecules that play a role in axonal growth and guidance.

Vertebrate orthologues of the Drosophila region-specific patterning gene teashirt.

In Drosophila the teashirt gene, coding for a zinc finger protein, is active in specific body parts for patterning. For example, Teashirt is required in the trunk (thorax and abdomen) tagmata of the embryo, parts of the intestine and the proximal parts of appendages. Here we report the isolation of vertebrate cDNAs related to teashirt. As in Drosophila, human and murine proteins possess three widely spaced zinc finger motifs. Additionally, we describe the expression patterns of the two murine genes. Both genes show regionalized patterns of expression, in the trunk, in the developing limbs and the gut.

Electromicroelution and microfingerprinting of radiolabeled immunoglobulins from sodium dodecyl sulfate-polyacrylamide gels.

An apparatus for electromicroelution from gel slices is described. After 20 min 70-90% of radiolabeled molecules can be extracted from the gel and recovered in 100 mul of buffer. The extracted molecules could be analysed in SDS-PAGE after reduction or could be split to perform microfingerprints (4 cm X 4 cm). This method has been used in several studies on membrane molecules (Fc gamma receptor, surface IgD, T cell receptor).

Insulin-specific T cell help to TNP-specific B cells requires activation via the antigen T cell receptor.

In the present study, the production of large numbers of insulin (Ins)-specific, H-2-restricted T helper (Th) cell clones is described. Among 148 clones analyzed, 121 clones had Th cell function, and these were divided into 75 Ins-specific Th cell clones and 52 autoreactive clones. The 148 clones were isolated from Ins-specific T cell lines produced by in vitro stimulation of T lymphocytes from (b X d)F1 mice immunized with Ins 7, 14, 32 or 56 days before. The following characteristics were tested with regard to the Th cell clones: restriction specificity and antigen requirement for optimal help or interleukin 2 (IL2) production. No differences in these characteristics were found among clones originating from day 7, 14, 32 or 56 T cell lines. A preference for H-2b as restriction element and an antigen concentration of about 0.01 microgram trinitrophenylated (TNP)-Ins/ml for optimal help were general traits. Optimal IL2 release is not yet obtained with 100 micrograms TNP-Ins/ml. Thus, the antigen requirement for optimal help and IL2 release differs by a factor 10(4) at least. Certain twice-cloned Th cell lines were tested for IL2 production when stimulated with anti-Thy-1 monoclonal antibodies (mAb). All clones analyzed were stimulated by mAb H155 but not with mAb H140 nor with mAb against Lyt-1, L3T4, LFA-1 or H-2K/D molecules. Therefore, we determined whether TNP-conjugated H155 mAb would mediate Th cell-B cell collaboration as well as TNP-Ins. The results with nine different Th cell clones and six different TNP-conjugated mAb used in a 10(7)-fold concentration range showed that Th cell clones have to be triggered via the T cell receptor for expression of helper function to B cells. Thus, though IL2 gene activation, synthesis or release apparently can be activated via at least two pathways: T cell receptor or Thy-1, it seems that activation of the genes responsible for synthesis and release of the helper factors, which ensure antigen-specific B cell proliferation and differentiation, needs Th cell-B contact mediated via the antigen-specific T cell receptor.

Putative conformation of mouse Fc gamma-receptor.

Three cell surface molecules (m.w. = 115,000, 90,000, and 70,000) binding to the Fc portion of complexed IgG have been isolated from the murine mastocytoma line P815. Various results suggested that the 90,000 and 70,000 dalton components are generated from the 115,00 dalton molecule by spontaneous proteolytic clevages and release of 23,000 dalton fragments. It was demonstrated that these cleavages occur during cell culture and not when freshly harvested mouse spleen cells are used as an Fc gamma R cell source. The survey of the Fc gamma R molecular forms obtained from P815 and spleen cells together with their reduction products led us to conclude that the mouse Fc gamma-receptor for complexed IgG is a single chain molecule (115,000 daltons) folded into five globular subunits (m.w. eta 23,000) linked by loose connecting regions accessible to proteolytic enzymes. Three of these subunits that compose the 70,000-dalton fragment are linked by di-sulfide bonds. Furthermore, a 140,000-dalton Fc gamma-binding molecule, not identified after cell surface labeling, could be detected after internal labeling. This component could be a cytoplasmic precursor of the Fc gamma R molecule. The structural model we present here might in addition shed some light on the discrepancy that appears through the various biochemical studies performed so far on Fc gamma-receptors.

Genetic chasing of T helper cell idiotype and allotype genes.

The present experiments were performed to study whether the genes responsible for the expression of T-idiotypes and allotypes could be mapped in relation to immunoglobulin (Ig) heavy chain V- and C-genes. Use was made of our antiserum 5936, which detects idiotypes in B6 and anti-B10.BR sera and on Lyt-1+, 2.3-B6 anti-B10.BR T-cell populations, and antiserum 6036, which detects allotypes on Lyt-1+, 2.3-B6 T cells, but which does not react against Ig. The reactivity of these antisera with T cells from (B6 X C3H.OH) X C3H.OH backcross mice and CBA-allotype congenic B6 mice was investigated because 5936 idiotypes and 6036 allotypes appeared to be associated with Igh-1b genes (B6) and not with Igh-1j genes (C3H.OH,CBA). Our results will show, first, that 5936 idiotypes on Lyt-1+, s.3-B6 anti-B10.BR T cells are synthesized by genes linked to Igh-1b allotype genes and they are situated either within Ig heavy chain V-genes or centromeric to them. Second, our results will show that 6036 allotypes on Lyt-1+, 2.3-B6 T cells are produced by genes also linked to Igh-1b-allotype genes, and the 6036 allotype genes are situated between Ig-VH and prealbumin genes.

Altered retinoic acid sensitivity and temporal expression of Hox genes in polycomb-M33-deficient mice.

The Polycomb group genes are required for the correct expression of the homeotic complex genes and segment specification during Drosophila embryogenesis and larval development. In mouse, inactivation studies of several Polycomb group genes indicate that they are also involved in Hox gene regulation. We have used our previously generated M33 mutants to study the function of M33, the mouse homologue of the Polycomb gene of Drosophila. In this paper, we show that in the absence of M33, the window of Hoxd4 retinoic acid (RA) responsiveness is opened earlier and that Hoxd11 gene expression is activated earlier in development This indicates that M33 antagonizes the RA pathway and has a function in the establishment of the early temporal sequence of activation of Hox genes. Despite the early activation, A-P boundaries are correct in later stages, indicating a separate control mechanism for early aspects of Hox regulation. This raises a number of interesting issues with respect to the roles of both Pc-G proteins and Hox regulatory mechanisms. We propose that a function of the M33 protein is to control the accessibility of retinoic acid response elements in the vicinity of Hox genes regulatory regions by direct or indirect mechanisms or both. This could provide a means for preventing ectopic transactivation early in development and be part of the molecular basis for temporal colinearity of Hox gene expression.

Differential exon usage involving an unusual splicing mechanism generates at least eight types of NCAM cDNA in mouse brain.

The murine neural cell adhesion molecule (NCAM) is known to exist in three isoforms of different size, NCAM-180, -140 and -120 coded for by four transcripts of 6.9, 6.1, 4.8 and 2.7 kb in length. Since the differences between these isoforms are due to alternative splicing in the coding region for the transmembrane and cytoplasmic domains, the extracellular, N-terminal portion of NCAM seemed to be shared by all three protein forms. Here we report that the coding region for N-terminal domains of NCAM also contains at least two sites of alternative splicing, termed alpha and pi. Short additional sequences of 3, 18 and 30 nt in length can be introduced at these sites, which are located in the membrane-proximal 'stem' between the Ig-like domains and the membrane attachment site and within the Ig-like domain IV, respectively. Proof for at least eight different mRNAs has been found by sequencing and S1 nuclease protection assays of selected independent cDNA clones, and Northern blot analyses. If most combination of the splice patterns identified so far in mouse brain occurred, 24 different mRNAs could be generated coding for 18 different proteins. The shortest extra-sequence found inserted at splice site alpha consisted only of the trinucleotide AAG, raising questions about the mechanism of this particular insertion.

On the molecular basis of T-helper-cell function. IV. B-lymphocyte-promotor factors: on their mode of action, biochemical nature and possible relationship to molecules involved in specific T-helper-cell activity.

Some further aspects of B-lymphocyte-promotor factor (B-LPF) activity have been studied. This activity was present in the supernatants of certain helper-T-cell lines, and it induced polyclonal activation of Ig+ B cells into Ig-secreting cells. It was found that B-LPF induced polyclonal, terminal B-cell differentiation (1) in T-cell- and macrophage-depleted spleen cell populations, (2) in both Lyb-5- and Lyb-5+ cells as well as in small and blast-like splenic B cells, and (3) in normal rather than memory B cells. B-LPF function was neither restricted to major histocompatibility complex gene products nor to immunoglobulin allotypes. B-LPF-like activity was also produced by some B-cell lymphomas/hybrids and by the P388-D1 macrophage line. B-LPF activity was found in three MW fractions: (I) greater than 180,000 (pI greater than 7.0 and 4.5-5.5); (II) 50,000-70,000 (pI greater than 7.0; 6.0-6.5, and 4.5-5.5); and (III) 10,000-15,000 (pI greater than 7.0). All three MW forms of B-LPF activity carried antiserum 6036-defined and AB-1.9.3 monoclonal antibody-defined determinants, and they reacted with chicken gammaglobulin (CGG)-Sepharose but not with human serum albumin-Sepharose. These data indicate that the three MW forms of B-LPF activity are associated/dissociated forms of a 10,000-15,000 MW form (subunit) rather than three different molecular species with B-LPF activity. A comparative study between antigen-specific helper factors and B-LPF was hampered by the finding that the helper-T-cell hybridomas used (e.g., T85-109-45/1) only produced B-LPF in our hands. Previously, it has been described that these helper-T-cell hybrids produced CGG-specific, I-Ak-restricted helper factors. However, one surprising observation was that B-LPF produced by both T85 hybrid cells and L12 T lymphoma cells was absorbed and could be eluted from CGG-Sepharose columns. The relationship of B-LPF to other nonspecific and apparently specific T-helper-cell products is discussed in particular in the light of the observations that many immunologically active molecules are built up from 10,000-12,000 molecular weight domain-like polypeptide structures.

The 5936 Ig-idiotype(s): genetic linkage to Ig-Ch Loci, T-cell dependence of synthesis and possible specificities.

5936-idiotype(s) are defined by a rabbit antiserum which was obtained by immunizing a mouse-immunoglobulin(MIg)-tolerant rabbit 5936 with B6 anti-CBA Ig from a single B6 mouse. These idiotypes were found on IgG1 molecules in sera from B6 mice immunized with cells carrying the IAk alloantigen, and they were associated with B6 Ig allotypes (Igh-1b). The particular interest of this idiotypic system is that antiserum 5936 also detects idiotypes (Id) on B6-T cells immunized in vitro with IAk alloantigen, and such T-cell Id were found to be associated with Ig allotypes. We attempted to show that the 5936-Id on Ig molecules were linked to Igh-1b allotypes by backcross analysis. In addition, it was investigated whether the 5936-Id response was a T-cell dependent response, and, if so whether there was any restriction in this T-B cell collaboration. Finally, we continued our studies on the specificity of 5936-Id+ Ig molecules. The present experiments show that 5936-Id are linked to Igh-1b allotypes and that the quantity of 5936-Id produced appears to be regulated by Ir genes in the H-2 complex. The synthesis of 5936-Id is T-cell dependent, and only Igh-1b allotype compatible T cells would collaborate with 5936-Id+ B-cell precursors. 5936-Id+ Ig molecules appeared not to have specificity against eco- and xeno-tropic viruses. However, monoclonal antibodies produced from B6-T-cell-reconstituted B6 nude mice may carry 5936-Id and have specificity for B6-T-cell membrane molecules. The relevance of these findings to the question of whether or not T and B cells display identical idiotypes are discussed.

Analysis of a fetal calf serum-induced T cell line. I. H-2 restricted growth and expression of membrane-bound and released molecules bearing 5936-idiotypic determinants.

It was previously established that a fetal calf serum-induced C57BL/6 T cell line that induces T and B cell differentiation could be kept proliferating in vitro only if cultured in the presence of irradiated syngeneic spleen cells and FCS. The present experiments were performed in order to investigate a) whether this cell line was a pure T cell line, b) whether the cells in this cell line (called line 12) were homogeneous with regard to Lyt phenotype, c) whether its growth was H-2 restricted, and d) whether line 12 cells reacted with our anti-idiotype (5936) and anti-T cell receptor allotype/isotype (6036) antisera. The results showed that line 12 consisted of T cells of Lyt 1+, 2.3- and phenotype. Its growth and proliferation was restricted to Kb and/or IAb alloantigen, and this phenomenon was observed with isolated Lyt 1+, 2.3- T cells. Line 12 cells reacted with both 5936 and 6036 antisera, and the positive cells were of Lyt 1+, 2.3- phenotype. Thus, our data indicate that Lyt 1+, 2.3- line 12 T cells interact with FCS and Kb/IAb alloantigen via receptors, which may bear 5936 and 6036 antisera-defined determinants. However, because these antisera only react with a subpopulation of Lyt 1+, 2.3- cells, proof that the same T cell has both MHC specificity and B cell idiotypic determinants will require further experimentation. 5936 and 6036 antisera-reactive molecules could be isolated from the supernatants of line 12 cells. Such molecules had characteristics similar to the 50,000 m.w. form of receptor molecules isolated from B6 anti-CBA T cell supernatants: a single chain polypeptide carrying both 5936 and 6036 antisera-defined determinants.