The neural restrictive silencer element can act as both a repressor and enhancer of L1 cell adhesion molecule gene expression during postnatal development

The cell adhesion molecule L1 mediates axonal guidance during neural development and mutations in its gene result in severe neurological defects. In previous studies, we identified the promoter for the L1 gene and showed that a neural restrictive silencer element (NRSE) was critical for preventing ectopic expression of L1 during early embryonic development. In the present study, we have investigated the role of the NRSE in the regulation of L1 expression during postnatal development. In gel mobility shift experiments, the NRSE formed DNA-protein complexes with nuclear extracts prepared from the brains of postnatal mice. To examine the influence of the NRSE on postnatal patterns of L1 expression in vivo, we compared the expression of two lacZ transgene constructs, one containing the native L1 gene regulatory sequences (L1lacZ) and another (L1lacZDeltaN) lacking the NRSE. Newborn mice carrying the L1lacZDeltaN showed enhanced beta-galactosidase expression relative to L1lacZ in the brain and ectopic expression in nonneural tissues. In contrast to L1lacZ mice, however, L1lacZDeltaN mice showed an unexpected loss, during postnatal development and in the adult, of beta-galactosidase expression in several neural structures, including the neural retina, cerebellum, cortex, striatum, and hippocampus. These data support the conclusion that the NRSE not only plays a role in the silencing of L1 expression in nonneural tissues during early development but also can function as a silencer and an enhancer of L1 expression in the nervous system of postnatal and adult animals.     

MEMD, a new cell adhesion molecule in metastasizing human melanoma cell lines, is identical to ALCAM (activated leukocyte cell adhesion molecule)

From a differential mRNA display comparing a non- and a highly metastasizing human melanoma cell line, we isolated and characterized memD. memD is preferentially expressed in the highly metastasizing melanoma cell lines of a larger panel. The encoded protein, MEMD, is identical to activated leukocyte cell adhesion molecule (ALCAM), a recently identified ligand of CD6. ALCAM is involved in homophylic (ALCAM-ALCAM) and heterophylic (ALCAM-CD6) cell adhesion interactions. We have studied MEMD/ALCAM cell-cell interactions between human melanoma cells. The expression of this cell adhesion molecule not only correlates with enhanced metastatic properties and with aggregational behavior of human melanoma cells as tested by FACS analysis, but transfection experiments also make clear that MEMD/ALCAM expression is essential for cell-cell interaction of the investigated human melanoma cells. As the melanoma cell lines analyzed are all CD6 negative, these results strongly suggest that MEMD/ALCAM is an adhesion molecule mediating homophylic clustering of melanoma cells. MEMD/ALCAM expression is not restricted to subsets of leukocytes and melanoma cells, it can also be found in healthy organs and in several other malignant tumor cell lines. Besides, MEMD/ALCAM is also expressed in cultured endothelial cells, pericytes and melanocytes, in xenografts derived from the radial and vertical growth phase and in 4 of 13 melanoma metastasis lesions. The potential role is discussed of MEMD/ALCAM mediated cell-cell interactions in migration of mobile cells (ie, activated leukocytes, metastasizing tumor cells) through tissues.     

Identification and characterization of the murine homologue of CD22, a B lymphocyte-restricted adhesion molecule

The human B lymphocyte-specific Ag, CD22, is a cell adhesion molecule expressed on the surface during a narrow window of B cell development, coincident with surface IgD. A ligand for CD22 has recently been identified on human T cells as the low molecular mass isoform of the leukocyte common Ag, CD45RO. CD22 has been reported to function in the regulation of both T and B cell activation in vitro. In this study, we report the isolation and expression of a molecular cDNA clone encoding the murine homologue of CD22, mCD22. Within their predicted protein sequences, murine and human sequences overall have 62% identity, which includes 18 of 20 extracellular cysteines and six of six cytoplasmic tyrosines. BHK cells transfected with mCD22 cDNA specifically adhere to resting and activated T lymphocytes and in addition bound activated, but not resting, B cells. Five Th clones were analyzed for their ability to adhere to mCD22; two Th0 clones and one Th1 clone bound CD22+ BHK transfectants, but not all T cell clones bound CD22+ cells: another Th1 clone and a Th2 clone did not. mCD22+ BHK transfectants were also specifically bound by the B cell-specific mAb, NIM-R6, demonstrating that this mAb is specific for murine CD22. Human cell lines expressing the counter-receptors for human CD22 were also examined for adhesion to the murine CD22 homologue; the epitope responsible for B cell adhesion to CD22 is conserved, whereas the T cell epitope binding to CD22 is not. The cDNA and mAb to murine CD22 will be useful for defining the in vivo function of CD22.     

Identification and characterization of glycosylation-dependent cell adhesion molecule 1-like protein expression in the ovine uterus

We cloned the dbl-1 gene, a C. elegans homolog of Drosophila decapentaplegic and vertebrate BMP genes. Loss-of-function mutations in dbl-1 cause markedly reduced body size and defective male copulatory structures. Conversely, dbl-1 overexpression causes markedly increased body size and partly complementary male tail phenotypes, indicating that DBL-1 acts as a dose-dependent regulator of these processes. Evidence from genetic interactions indicates that these effects are mediated by a Smad signaling pathway, for which DBL-1 is a previously unidentified ligand. Our study of the dbl-1 expression pattern suggests a role for neuronal cells in global size regulation as well as male tail patterning.     

Identification of the gene encoding scHelI, a DNA helicase from Saccharomyces cerevisiae

The gene encoding scHelI, a previously characterized DNA helicase from Saccharomyces cerevisiae, has been identified as YER176w, an open reading frame on chromosome V. The gene has been named HEL1 to indicate the DNA helicase activity of the gene product. HEL1 was identified by screening a lambda gt11 yeast protein expression library with antiserum to purified scHelI. Several independent immunopositive clones were isolated and shown to contain portions of HEL1 either by sequencing or by hybridization to a probe containing HEL1 sequences. The HEL1 open reading frame includes the seven conserved helicase motifs, consistent with the DNA helicase activity of scHelI, and the predicted size of the protein is in agreement with the size of purified scHelI. Partially purified cellular extracts from a hel1 deletion mutant strain did not contain scHelI activity. Homology searches revealed protein sequence homology between HEL1 and two previously identified and biochemically characterized yeast helicases, encoded by the DNA2 and UPF1 genes. Haploid hel1 deletion strains were constructed and shown to be viable with growth rates equivalent to those of parental strains. These strains did not differ from the parental strains in ultraviolet light sensitivity or the generation of petite colonies. Furthermore, these haploid deletion strains were capable for mating, the resultant diploid homozygous mutants were viable, capable of sporulation, and the spores displayed no reduction in viability.     

Altered secondary myogenesis in transgenic animals expressing the neural cell adhesion molecule under the control of a skeletal muscle alpha-actin promoter

The majority of skeletal muscle fibers are generated through the process of secondary myogenesis. Cell adhesion molecules such as NCAM are thought to be intricately involved in the cell-cell interactions between developing secondary and primary myotubes. During secondary myogenesis, the expression of NCAM in skeletal muscle is under strict spatial and temporal control. To investigate the role of NCAM in the regulation of primary-secondary myotube interactions and muscle fusion in vivo, we have examined muscle development in transgenic mice expressing the 125-kD muscle-specific, glycosylphosphatidylinositol-anchored isoform of human NCAM, under the control of a human skeletal muscle alpha-actin promoter that is active from about embryonic day 15 onward. Analysis of developing muscle from transgenic animals revealed a significantly lower number of myofibers encased by basal lamina at postnatal day 1 compared with nontransgenic littermates, although the total number of developing myofibers was similar. An increase in muscle fiber size and decreased numbers of VCAM-1-positive secondary myoblasts at postnatal day 1 was also found, indicating enhanced secondary myoblast fusion in the transgenic animals. There was also a significant decrease in myofiber number but no increase in overall muscle size in adult transgenic animals; other measurements such as the number of nuclei per fiber and the size of individual muscle fibers were significantly increased, again suggesting increased secondary myoblast fusion. Thus the level of NCAM in the sarcolemma is a key regulator of cell-cell interactions occurring during secondary myogenesis in vivo and fulfills the prediction derived from transfection studies in vitro that the 125-kD NCAM isoform can enhance myoblast fusion.     

Spatio-temporal diversity in the microenvironments for neural cell adhesion molecule, neural cell adhesion molecule-polysialic acid, and L1-cell adhesion molecule expression by sensory neurons and their targets during cochleo-vestibular innervation

Sixteen phases in the microenvironments were defined for the structural development and innervation of the cochleo-vestibular ganglion and its targets. In each phase the cell adhesion molecules, neural cell adhesion molecule, neural cell adhesion molecule-polysialic acid, and L1-cell adhesion molecule, were expressed differentially by cochleo-vestibular ganglion cells, their precursors, and the target cells on which they synapse. Detected by immunocytochemistry in staged chicken embryos, in the otocyst, neural cell adhesion molecule, but not L1-cell adhesion molecule, was localized to the ganglion and hair cell precursors. Ganglionic precursors, migrating from the otocyst, only weakly expressed neural cell adhesion molecule. Epithelial hair cell precursors, remaining in the otocyst, expressed neural cell adhesion molecule, but not L1-cell adhesion molecule. Post-migratory ganglion cell processes expressed both molecules in all stages. The cell adhesion molecules were most heavily expressed by axons penetrating the otic epithelium and accumulated in large amounts in the basal lamina. In the basilar papilla (cochlea), cell adhesion molecule expression followed the innervation gradient. Neural cell adhesion molecule and L1 were heavily concentrated on axonal endings peripherally and centrally. In the rhombencephalon, primitive epithelial cells expressed neural cell adhesion molecule, but not L1-cell adhesion molecule, except in the floorplate. The neuroblasts and their axons expressed L1-cell adhesion molecule, but not neural cell adhesion molecule, when they began to migrate and form the dorsal commissure. There was a stage-dependent, differential distribution of the cell adhesion molecules in the floorplate. Commissural axons expressed both cell adhesion molecules, but their polysialic acid disappeared within the floorplate at later stages. In conclusion, the cell adhesion molecules are expressed by the same cells at different times and places during their development. They are positioned to play different roles in migration, target penetration, and synapse formation by sensory neurons. A multiphasic model provides a morphological basis for experimental analyses of the molecules critical for the changing roles of the microenvironment in neuronal specification.     

Cytoplasmic tail regulates the intercellular adhesion function of the epithelial cell adhesion molecule

Ep-CAM, an epithelium-specific cell-cell adhesion molecule (CAM) not structurally related to the major families of CAMs, contains a cytoplasmic domain of 26 amino acids. The chemical disruption of the actin microfilaments, but not of the microtubuli or intermediate filaments, affected the localization of Ep-CAM at the cell-cell boundaries, suggesting that the molecule interacts with the actin-based cytoskeleton. Mutated forms of Ep-CAM were generated with the cytoplasmic domain truncated at various lengths. All of the mutants were transported to the cell surface in the transfectants; however, the mutant lacking the complete cytoplasmic domain was not able to localize to the cell-cell boundaries, in contrast to mutants with partial deletions. Both the disruption of the actin microfilaments and a complete truncation of the cytoplasmic tail strongly affected the ability of Ep-CAM to mediate aggregation of L cells. The capability of direct aggregation was reduced for the partially truncated mutants but remained cytochalasin D sensitive. The tail truncation did not affect the ability of the transfectants to adhere to solid-phase-adsorbed Ep-CAM, suggesting that the ability to form stable adhesions and not the ligand specificity of the molecule was affected by the truncation. The formation of intercellular adhesions mediated by Ep-CAM induced a redistribution to the cell-cell boundaries of alpha-actinin, but not of vinculin, talin, filamin, spectrin, or catenins. Coprecipitation demonstrated direct association of Ep-CAM with alpha-actinin. Binding of alpha-actinin to purified mutated and wild-type Ep-CAMs and to peptides representing different domains of the cytoplasmic tail of Ep-CAM demonstrates two binding sites for alpha-actinin at positions 289 to 296 and 304 to 314 of the amino acid sequence. The results demonstrate that the cytoplasmic domain of Ep-CAM regulates the adhesion function of the molecule through interaction with the actin cytoskeleton via alpha-actinin.     

Identification of a poxvirus gene encoding a uracil DNA glycosylase

An open reading frame, BamHI D6R, from the central highly conserved region of the Shope fibroma virus (SFV) genome was sequenced and found to have significant homology to that of uracil DNA glycosylases from a number of organisms. Uracil DNA glycosylase catalyzes the initial step in the repair pathway that removes potentially mutagenic uracil from duplex DNA. The D6R polypeptide was expressed in reticulocyte lysates programmed with RNA transcribed from an expression vector containing the T7 RNA polymerase promoter. A highly specific ethidium bromide fluorescence assay of the in vitro translation product determined that the encoded protein does indeed possess uracil DNA glycosylase activity. Open reading frames from other poxviruses, including vaccinia virus (HindIII D4R) and fowlpox (D4), are highly homologous to D6R of SFV and are predicted to encode uracil DNA glycosylases. Identification of the SFV uracil DNA glycosylase provides evidence that this poxviral protein is involved in the repair of the viral DNA genome. Since this enzyme performs only the initial step required for the removal of uracil from DNA, creating an apyrimidinic site, we suggest that other, possibly virus-encoded, repair activities must be present in the cytoplasm of infected cells to complete the uracil excision repair pathway.