Ig gene rearrangement and expression in the progeny of B-cell progenitors in the course of clonal expansion in bone marrow cultures.

In cultures of murine bone marrow cells colonies of 10(3)-10(4) cells were identified which consisted to a large part of pre-B and B cells. Cell mixing experiments with genetically marked cells indicated that each colony is derived from a single progenitor cell not yet committed to the expression of either IgH locus. A concanavalin-A-mediated electrofusion method allowed us to rescue and amplify individual cells from a given colony by hybridization with X63.Ag8.653 cells. The molecular analysis of 12 such hybridomas revealed that all IgH loci were rearranged into DJH or productive or nonproductive VHDJH complexes. Most kappa and all lambda light chain loci were in germline configuration. Kappa chain expression was only seen in heavy (mu) chain expressing hybridomas. Hybridomas from a given colony were heterogeneous in terms of DJH and VHDJH rearrangements and in no cell was more than one productive VHDJH complex detected. None of the productive VHDJH complexes contained a VH gene of group 1 (J558), the largest VH gene family with about half of the VH genes. This is in marked contrast to VH gene usage in splenic B cells.     

The accumulation of mature RNA for the Xenopus laevis ribosomal protein L1 is controlled at the level of splicing and turnover of the precursor RNA.

A specific control regulates, at the level of RNA splicing, the expression of the L1 ribosomal protein gene in Xenopus laevis. Under particular conditions, which can be summarized as an excess of free L1 protein, a precursor RNA which still contains two of the nine introns of the L1 gene accumulates. In addition to the splicing block the two intron regions undergo specific endonucleolytic cleavages which produce abortive truncated molecules. The accumulation of mature L1 RNA therefore results from the regulation of the nuclear stability of its precursor RNA. We propose that a block to splicing can permit the attack of specific intron regions by nucleases which destabilize the pre-mRNA in the nucleus. Therefore the efficiency of splicing could indirectly control the stability of the pre-mRNA.     

Characterization of the cell adhesion molecules L1, N-CAM and J1 in the mouse intestine.

To gain insight into the cellular and molecular mechanisms underlying epithelial cell surface interactions in the adult mouse intestine, we have characterized the cell adhesion molecules L1, N-CAM and J1 by immunocytological, biochemical and cell biological methods. Whereas N-CAM and J1 expression was found to be confined to the mesenchymal and neuroectodermally-derived parts of the intestine, L1 was localized in the proliferating epithelial progenitor cells of crypts, but not in the more differentiated epithelial cells of villi. L1 was detected in crypt cells by Western blot analysis in the molecular forms characteristic of peripheral neural cells, with apparent mol. wts of 230, 180 and 150 kd. Aggregation of single, enriched crypt, but not villus cells, was strongly inhibited in the presence of Fab fragments of polyclonal L1 antibodies. These observations show that L1 is not confined to the nervous system and that it may play a functional role in the histogenesis of the intestine in the adult animal.     

(dC-dA)n.(dG-dT)n sequences have evolutionarily conserved chromosomal locations in Drosophila with implications for roles in chromosome structure and function.

In situ hybridization of (dC-dA)n.(dG-dT)n to the polytene chromosomes of Drosophila melanogaster reveals a clearly non-random distribution of chromosomal sites for this sequence. Sites are distributed over most euchromatic regions but the density of sites along the X chromosome is significantly higher than the density over the autosomes. All autosomes show approximately equal levels of hybridization except chromosome 4 which has no detectable stretches of (dC-dA)n.(dG-dT)n. Another striking feature is the lack of hybridization of the beta-heterochromatin of the chromocenter. The specific sites are conserved between different strains of D. melanogaster. The same overall chromosomal pattern of hybridization is seen for the other Drosophila species studied, including D. simulans, a sibling species with a much lower content of middle repetitive DNA, and D. virilis, a distantly related species. The evolutionary conservation of the distribution of (dC-dA)n.(dG-dT)n suggests that these sequences are of functional importance. The distribution patterns seen for D. pseudoobscura and D. miranda raise interesting speculations about function. In these species a chromosome equivalent to an autosomal arm of D. melanogaster has been translocated onto the X chromosome and acquired dosage compensation. In each species the new arm of the X also has a higher density of (dC-dA)n.(dG-dT)n similar to that seen on other X chromosomes. In addition to correlations with dosage compensation, the depletion of (dC-dA)n.(dG-dT)n in beta-heterochromatin and chromosome 4 may also be related to the fact that these regions do not normally undergo meiotic recombination.     

Expression of retroviral vectors in transgenic mice obtained by embryo infection.

Pre-implantation embryos were infected with the retroviral vector MMCV-neo, which carries the neomycin resistance (neo) gene and the v-myc gene. Three transgenic substrains (M-TKneo 1-3) were derived which stably transmit a single intact copy of the vector. In all of the substrains, expression of the neo gene from the internal thymidine kinase (TK) promoter was detected, with two of the substrains expressing the gene in all tissues analysed. In the third substrain, the vector had integrated on the X chromosome and neo expression varied between different tissues. A second series of transgenic mice were obtained with the retroviral vector SAX, in which the human adenosine deaminase cDNA (ADA) is under the control of an internal SV40 promoter. Four substrains (M-SAX 1-4) were analysed; however, no expression of the ADA cDNA was detected. In all mice, no expression was found of the genes under the control of the viral 5' long terminal repeats (LTRs). In the M-TKneo substrains the vector was hypomethylated irrespective of its expression whereas in the M-SAX mice the vector was hypermethylated. These results demonstrate for the first time that the TK promoter can apparently express a gene in all tissues of adult mice and that retroviral vectors with internal promoters may provide an alternative to DNA injection for the efficient expression of genes in transgenic mice.     

Extracellular release of colicin A is non-specific.

The possible involvement of topogenic export sequences within the colicin A polypeptide chain has been investigated. Different constructs have been made using various techniques to introduce deletions in the central and NH2-terminal regions of colicin A. Together, these deletions span the region from amino acid 15 to the end of the protein. None of these regions was found to be required for extracellular release or had any effect on the efficiency of this process. By inserting a termination codon, a Shine-Dalgarno sequence and an initiation codon into the gene for colicin A, the NH2-terminal and central plus COOH-terminal domains could be demonstrated to be released to the same extent when produced as separate polypeptides as when produced as linked ones. The introduction into the COOH-terminal domain of mutations promoting cytoplasmic aggregation had no effect on the secretion of the NH2-terminal polypeptide. These results demonstrated that no specific interaction between the NH2- and COOH-terminal regions of the colicin A polypeptide chain is involved in the release of colicin A. We are led to conclude that there is no topogenic export signal in the polypeptide chain of colicin A involved in the release mechanism. Thus the process is non-specific with respect to the colicin itself and depends solely on the expression of the colicin A lysis protein (Cavard et al., 1985, 1987). The expression of the protein causes the release of not only the colicin but also many other cellular proteins, including beta-lactamase, EF-Tu, and chloramphenicol acetyltransferase.     

Internalization and degradation of peptides of the bombesin family in Swiss 3T3 cells occurs without ligand-induced receptor down-regulation.

The binding of [125I]gastrin releasing peptide ([125I]GRP) to Swiss 3T3 cells at 37 degrees C increases rapidly, reaching a maximum after 30 min and decreasing afterwards. The decrease in cell-associated radioactivity at this temperature is accompanied by extensive degradation of the labelled peptide. At 4 degrees C equilibrium binding is achieved after 6 h and [125I]GRP degradation is markedly inhibited. Extraction of surface-bound ligand at low pH demonstrates that the iodinated peptide is internalized within minutes after addition to 3T3 cells at 37 degrees C. The rate of internalization is strikingly temperature-dependent and is virtually abolished at 4 degrees C. In addition, lysomotropic agents including chloroquine increase the cell-associated radioactivity in cells incubated with [125I]GRP. The binding of [125I]GRP to Swiss 3T3 cells was not affected by pretreatment for up to 24 h with either GRP or bombesin at mitogenic concentrations. Furthermore, pretreatment with GRP did not reduce the affinity labelling of a Mr 75,000-85,000 surface protein recently identified as a putative receptor for bombesin-like peptides. These results demonstrate that while peptides of the bombesin family are rapidly internalized and degraded by Swiss 3T3 cells, the cell surface receptors for these molecules are not down-regulated.     

Organization of multispecific DNA methyltransferases encoded by temperate Bacillus subtilis phages.

B. subtilis phage rho 11s codes for a multispecific DNA methyltransferase (Mtase) which methylates cytosine within the sequences GGCC and GAGCTC. The Mtase gene of rho 11s was isolated and sequenced. It has 1509 bp, corresponding to 503 amino acids (aa). The enzyme's Mr of 57.2 kd predicted from the nucleotide sequence was verified by direct Mr determinations of the Mtase. A comparison of the aa sequence of the rho 11s Mtase with those of related phages SPR and phi 3%, which differ in their methylation potential, revealed generalities in the building plan of such enzymes. At least 70% of the aa of each enzyme are contained in two regions of 243 and 109 aa at the N and C termini respectively, which are highly conserved among the three enzymes. In each enzyme, variable sequences separate the conserved regions. Variability is generated through the single or multiple use of related and unrelated sequence motifs. We propose that the recognition of those DNA target sequences, which are unique for each of the three enzymes, is determined by these variable regions. Evolutionary relationships between the three enzymes are discussed.