SCIP: a glial POU domain gene regulated by cyclic AMP

We have isolated cDNA clones encoding SCIP, a POU domain gene expressed by myelin-forming glial of the central and peripheral nervous systems. In purified Schwann cells cultured in the absence of neurons, expression of SCIP is suppressed. This suppression is relieved by cAMP, and induction of SCIP mRNA by this second messenger precedes cAMP induction of myelin-specific genes. Similarly, SCIP expression in vivo precedes full expression of myelin-specific genes in developing oligodendrocytes and Schwann cells. The sequence of the SCIP POU domain is identical to that of Tst-1, a recently identified member of a family of POU domain genes expressed by restricted subsets of neurons. Our results demonstrate that SCIP is also expressed by myelin-forming glia and suggest that it plays a central role in the progressive determination of these cells and their commitment to myelination.     

The octamer binding factor Oct6: cDNA cloning and expression in early embryonic cells.

We have cloned a cDNA encoding a novel octamer binding factor Oct6 that is expressed in undifferentiated ES cells. Expression of the Oct6 gene is downregulated upon differentiation of these cells by aggregate formation. Furthermore the gene is transiently up regulated during retinoic acid induced differentiation of P19 EC cells, reaching maximum levels of expression one day after RA addition. Sequence analysis of the cDNA encoding the Oct6 protein indicated that the Oct6 gene is a member of the POU-HOMEO domain gene family. The gene expresses a 3 kb mRNA encoding a 449 amino acid protein with an apparent molecular weight of 45 kD. The sequence of the Oct6 POU domain is identical to that of the rat SCIP (Tst-1) gene. The Oct6 expression pattern suggests a role for this DNA binding protein in neurogenesis as well as early embryogenesis.     

Isolation of a full-length mouse cDNA clone coding for an immunologically distinct p53 molecule.

Transfection of a cloned p53 gene into a p53 nonproducer Abelson murine leukemia virus-transformed cell line, L12, reconstituted p53 expression. The protein expressed in these cells was indistinguishable from that naturally expressed in p53 producer tumor cells. Conversely, p53 protein expressed in L12-derived clones that were established by transfection with a full-length p53 cDNA clone (pM8) exhibited a discrete immunological form. Immunoprecipitation of p53 with a panel of monoclonal anti-p53 antibodies showed that L12-derived clones that were transfected with the genomic p53 clone contained the same antigenic determinants as those found in the p53 protein expressed in tumor cells. These p53 proteins bound all monoclonal antibody types as well as the polyclonal anti-p53 tested. However, L12-derived clones established by transfection of the p53 cDNA clone (pM8) expressed a p53 protein that bound the RA3-2C2 and PAb200.47 anti-p53 monoclonal antibodies as well as polyclonal anti-p53 serum but totally lacked the antigenic receptor for the PAb122 and PAb421 monoclonal antibodies. The p53 proteins expressed by either genomic or cDNA p53 clones exhibited the same apparent molecular sizes and identical partial peptide maps. We suggest that transfection of the p53 gene induced expression of the entire group of the possible mRNA species, whereas cloned p53 cDNA (pM8) represented a single mRNA molecule that codes for a discrete species of p53 protein.     

Sequence of the human factor VIII-associated gene is conserved in mouse.

cDNA and genomic clones corresponding to the human factor VIII-associated gene (F8A) were isolated from mouse cDNA and F8A-enriched genomic libraries. The sequences of these clones revealed an intronless gene coding for 380 amino acids, with 85% identity to the predicted human sequence. The single murine gene copy is genetically linked to factor VIII, but appears to lie outside the factor VIII gene by physical mapping. Like the human gene, the mouse F8A gene is highly expressed in a wide variety of tissues. This evolutionary comparison has helped to clarify the derived amino acid sequence in the human and strongly supports the hypothesis that the F8A gene encodes a protein.     

Structure and expression of the human polymorphic epithelial mucin gene: an expressed VNTR unit

The human polymorphic epithelial mucin (PEM) is expressed apically by glandular epithelium and by the carcinomas that develop from these tissues. Previously isolated cDNA clones revealed that the core protein contained a large domain consisting of variable numbers of 60 bp tandem repeats (TR), making it an expressed minisatellite. We now report the full genomic sequence of the PEM gene, including 803 bp of 5' flanking sequence. The gene is composed of 7 exons and varies in size from approximately 4 to approximately 7 kb, depending on the number of tandem repeats in exon 2. Expression of PEM was obtained from a genomic clone in an Epstein-Barr virus based vector, after transfection into a human epithelial cell line, indicating the presence of effective regulatory sequences in this clone.     

Growth Arrest and Spontaneous Differentiation Are Initiated Through an Autocrine Loop in Clonally Derived Schwann Cells by α1-Procollagen I C-Propeptide

Schwann cells cloned from rat sciatic nerve survive and display self-induced growth suppression, or undergo spontaneous apoptosis, on long-term serum-free subconfluent culture. Strain SCL4.1/F7 sustained the capacity to growth arrest for up to 40 generations. A soluble activity transmitted between neighbouring cells of this strain suppresses DNA synthesis within three cell cycles. Autocrine Schwann cell growth-inhibitory factor (SGIF) operates during the G1 phase of the cell cycle, overcomes the mitogenic action of Schwann cell/serum-associated (platelet-derived growth factor-BB) and axon-associated (axolemma-enriched fraction) stimuli in serum-free conditions, and suppresses DNA synthesis in sciatic nerve Schwann cell cultures in a stage-specific manner. A 35-kDa protein with N-terminal sequence and approximate molecular mass of the C-propeptide of rat alpha1-procollagen I makes a major contribution to SGIF. Growth suppression in the SCL4.1/F7 strain is mediated by the ras/extracellular signal-regulated kinase pathway, is accompanied by down-regulation of erbB2/erbB3 and of tetraethylammonium-sensitive K+ currents, and is followed by transition of cells within 5-10 days from O4+, p75 nerve growth factor receptor (p75NGF-R)+, glial fibrillary acidic protein (GFAP)+ to O4+, p75NGF-R-, GFAP-, periaxin+ phenotypes. Oct-6/SCIP mRNA is present in both proliferating and growth-arrested SCL4.1/F7 cells. These results demonstrate an autocrine/ paracrine loop for the growth arrest of clonally derived Schwann cells in the absence of axons linked in part to the metabolism of collagen. Schwann cells thus appear to self-regulate growth in a negative as well as a positive direction through characterized molecular mechanisms and signal pathways.     

Bacterial and host-derived cationic proteins bind alpha2-laminins and enhance Mycobacterium leprae attachment to human Schwann cells

It has recently been demonstrated that laminin alpha2 chains present on the surface of Schwann cells are involved in the process of attachment of Mycobacterium leprae to these cells. In this study, a protein in the M. leprae cell wall that was found to be capable of binding alpha2-containing laminins (merosin) was isolated and characterized. The M. leprae laminin-binding protein was identified as a 21-kDa histone-like protein (Hlp), a highly conserved cationic protein present in other species of mycobacteria. The gene that encodes this protein was PCR amplified, cloned, and expressed, and the recombinant protein was shown to bind alpha2-laminins. More significantly, when added exogenously, Hlp was able to greatly enhance the attachment of mycobacteria to ST88-14 human Schwann cells. The capacity to bind alpha2-laminins and to enhance mycobacterial adherence to Schwann cells was also found in other cationic proteins such as host-derived histones. Moreover, mutation in the hlp gene was shown not to affect the capacity of mycobacteria to bind to ST88-14 cells, suggesting that alternative adhesins and/or pathways might be used by mycobacteria during the process of adherence to Schwann cells. The potential role of Hlp as a fortuitous virulence factor contributing to the pathogenesis of M. leprae-mediated nerve damage is discussed.     

Mouse ferritin H multigene family is polymorphic and contains a single multiallelic functional gene located on chromosome 19.

Multiple ferritin H subunit sequences are present in the genome of higher vertebrates, but it is not yet known with certainty if more than one is expressed. In this paper, we provide evidence that there is only one functional ferritin H gene in the mouse. We screened a mouse genomic library using a mouse ferritin H cDNA as a probe and characterized five clones. These genomic clones proved to contain three pseudogenes and two allelic forms of a unique functional gene. These two alleles differed by only two point mutations in the promoter and three in the first intron and by a 31-bp insertion in the first intron. They were equally expressed when transiently transfected in HeLa cells. These five genomic clones account for all the bands observed on a Southern blot of mouse genomic DNA hybridized with a ferritin H cDNA, and these bands present a restriction fragment length polymorphism between various representatives of the genus Mus. Using a DNA panel prepared from the backcross progeny (C57BL/6 X Mus spretus)F1 X C57BL/6, we localized the functional ferritin H gene (Fth) in region B of mouse chromosome 19 and established cen-Ly-1-Fth-Pax-2 as the most likely gene order, thus defining a conserved syntenic fragment with human chromosome 11q.     

Structure and expression of a Manduca sexta larval cuticle gene homologous to Drosophila cuticle genes

A genomic clone was isolated from the tobacco hornworm, Manduca sexta, by virtue of its similarity to a Drosophila larval cuticle gene. RNA analysis shows that this clone, B311, is expressed at times appropriate for a larval cuticle gene. Hybrid-selection experiments using B311 DNA show that it encodes a 14 x 10(3) Mr protein, LCP-14, which is precipitated by an antiserum to Manduca larval cuticle. We have sequenced both genomic and cDNA clones for the LCP-14 gene. A conceptual translation of the cDNA sequence shows that the LCP-14 protein is similar not only to another Manduca cuticle protein, but also to Drosophila, Sarcophaga and Hyalophora cecropia cuticle proteins. Since these proteins are found in flexible cuticle and have similar sequences, we conclude they are encoded by homologous genes.     

Molecular and genetic analysis of an embryonic gene, DC 8, from Daucus carota L

Summary To understand the morphogenetic and physiological processes occurring during plant embryogenesis, we isolated cDNA clones homologous to genes preferentially expressed during somatic embryogenesis. One of these cDNA clones detected an embryo-specific mRNA species with a corresponding protein of 66 kDa. The expression pattern of the mRNA is similar between somatic and zygotic embryos of carrots. To characterize the gene encoding this mRNA, we isolated the corresponding genomic clones. Molecular analysis of the DNA from several haploid and diploid carrots showed that the mRNA was encoded by a single copy gene, named DC 8. DNA sequence analysis showed that the gene consisted of three exons and coded for a hydrophilic protein with a central region composed of 17 repeats. At the NH₂-terminus no typical signal sequence was found. Immunocytochemical analysis localized the protein primarily in the vacuoles and protein bodies of zygotic embryos; the cytoplasm showed some antibody staining. The protein was also found in cell walls of endosperm tissue. The amount of DC 8 protein was too low for it to be categorized as a seed storage protein; its role in embryogenesis remains to be determined.     

Isolation of rat Schwann cell lines: use of SV40 T antigen gene regulated by synthetic metallothionein promoters

Synthetic promoter elements from the mouse metallothionein-I promoter controlling the expression of SV40 T antigen have been tested for their efficacy in cloning rat Schwann cell lines that retained the characteristic properties of these cells and could be passed in culture indefinitely. The constructed promoters contained either four (MT4) or five (MT5) copies of a metal regulatory element 5' to the CAAT and TATA elements from the HSV-1 thymidine kinase gene. Characterization of these promoters in transient expression assays and transformation assays showed that both MT5 and MT4 were approximately 10-fold and 15-fold, respectively, weaker than the wild-type MT-I promoter in the presence of heavy metal inducer. However, in the absence of inducer, the basal activity of both MT5 and MT4 was barely detectable and much lower than that of MT-I. Schwann cells were transfected with plasmids containing the SV40 T antigen gene under the control of the different metallothionein promoters and cell lines were established from each. Only with the MT5 and MT4 promoters were lines obtained that resembled secondary Schwann cells in culture in their morphology, generation time, and demonstration of contact inhibition. In the presence of zinc, the expression of T antigen in the lines derived with MT5 and MT4 was about 10-fold lower than that derived with MT-I. On removal of the inducer this level was reduced, and in one cell line T antigen was undetectable. Concomitant with the reduction in T antigen expression there was an increased expression of Po, a protein specific to myelin-forming Schwann cells, and a decreased expression of glial fibrillary acidic protein, a protein expressed only in nonmyelin-forming Schwann cells. These cell lines, therefore, closely resemble untransfected Schwann cells in culture.