Lano, a novel LAP protein directly connected to MAGUK proteins in epithelial cells.

Protein networks asymetrically distributed to basolateral and apical epithelial membranes maintain cell polarity and homeostasis of epithelial tissues. Genetic studies in non-vertebrates assigned two families of basolateral proteins, MAGUK (membrane-associated and guanylate kinase) and LAP (leucine-rich repeats and PDZ) proteins, to a common pathway crucial for the epithelial architecture and acting as a gatekeeper to malignancy. In mammals, three LAP proteins have been described, Densin-180, Erbin, and hScribble. Here, we identify a protein called Lano (LAP and no PDZ) only present in vertebrates and presenting strong identities with LAP proteins. Despite the lack of PDZ domain, Lano is located at the basolateral side of epithelial cells in a similar manner to Erbin and hScribble. Using in vitro and in vivo experiments, we demonstrate that Lano directly interacts with the PDZ domains of MAGUK proteins, including hDLG (human disc large), in epithelial cells. A second pool of Lano is complexed to Erbin. These LAP-MAGUK protein complexes coexist at the basolateral side of epithelial cells. We provide evidence for a direct interaction between LAP and MAGUK proteins, and we propose that various LAP-MAGUK networks targeted to the basolateral side of epithelial cells participate to homeostasis of epithelial tissues and tumor growth.

A sequence based computational identification of a Drosophila developmentally regulated TATA-less RNA polymerase II promoter and its experimental validation.

Many RNA polymerase II promoters lack the characteristic TATA box sequence located -25/-30 nucleotides upstream from the transcription start. In Drosophila, half of the promoters identified so far are TATA-deficient. The yemanuclein-alpha gene whose promoter activity is restricted to oogenesis, falls in this class. A number of upstream and downstream promoter elements have been identified for some TATA-less promoters. The yem-alpha promoter contains none of the consensus elements identified so far. Our work was based on the assumption that the physical parameters of the DNA could be used to predict the location of the yem-alpha promoter. A sequence based computational analysis allowed us to determine the characteristic changes of DNA curvature and helix stability in the presumptive regulatory region. Our experimental data were in good agreement with the computational analysis. We have started to investigate the general value of this approach by analyzing other promoters.

Implication of a 5' coding sequence in targeting maternal mRNA to the Drosophila oocyte.

Early accumulation of maternal mRNA in one of the cells of the cluster of 16 cystocytes is a critical event in the determination of the Drosophila oocyte. A number of developmentally important mRNAs have been shown to accumulate in the early oocyte. We report here the early expression of the yemanuclein-alpha (yem-alpha) transcript, its accumulation in the germarial oocyte and its dynamic localization in the growing oocyte. We have investigated the mechanisms involved in these processes. Microtubules are likely to be involved in both transport and localization as was shown for other maternal transcripts which behave similarly. However, unlike all the cases reported so far, transport and localization are not dependent on 3'UTR sequences. We show that the 5' coding sequence is necessary for the early accumulation of yem-alpha RNA in the oocyte and for its localization pattern during oogenesis.

The mouse NCAM gene displays a biphasic expression pattern during neural tube development.

The neural cell adhesion molecule (NCAM) is one of the most abundant cell adhesion molecules expressed in vertebrates and it is thought to play important roles as a regulator of morphogenetic processes, but little is known of its expression pattern in mammalian embryos. In this study, we have examined the developmental profile of NCAM gene expression in mouse embryos from gestational day 7.5 to 12.5, focusing on the developing neural tube. NCAM transcripts were first detected around day 8.5 in the somites and the forming neural tube. At this stage, NCAM transcripts were expressed in the neuroepithelium throughout the width of the neural groove and tube up to a rostral boundary within the hindbrain, whereas NCAM mRNA levels were very low or undetectable in the neuroepithelium of the head region. The positional restriction of NCAM expression was confirmed by immunohistochemistry at the protein, and by polymerase chain reaction analysis at the RNA level. Expression in the neuroepithelium was transient as the level of NCAM transcripts declined in the germinal layer beyond day 8.5. By day 9.5, strong NCAM expression had appeared on the earliest postmitotic neurones along the entire neuraxis, and this pattern of expression in all regions with differentiating neurones was maintained until day 12.5. We conclude that NCAM expression in the neural tube occurs in two spatiotemporal distinct waves: a first wave in the proliferating neuroepithelium showing positional dependence along the rostrocaudal axis, and a second wave on essentially all neurones that have become postmitotic.

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.

Molecular modelling of the immunoglobulin-like domains of the neural cell adhesion molecule (NCAM): implications for the positioning of functionally important sugar side chains.

The neural cell adhesion molecule (NCAM) is thought to mediate cell-cell adhesion by a homophilic mechanism involving binding sites located in the N-terminal region of the protein. This region of the molecule consists of five domains that are homologous to each other and share conserved residues with immunoglobulin domains. We report here secondary structure predictions for the five NCAM domains and three-dimensional models for two of them. The results are entirely consistent with an immunoglobulin-like folding of the NCAM domains into seven strands forming two beta-sheets. NCAM-NCAM binding may thus be analogous to the pairwise associations of immunoglobulin constant domains, which are involved in dimer formation. Insertions and deletions are located mostly in beta-turn regions. Two alpha-helical regions in the third and fourth domain are predicted with high probability. NCAM bears two kinds of functionally important sugar side chains, sialic acid polymers in the fifth domain, which modulate NCAM binding, and the L2 moiety, which is involved in cell adhesion and can be assigned to the third domain. Three-dimensional modelling of the corresponding domains indicates that two of the three sites for N-linked glycosylation in the fifth and the single site in the third domain are located on the face of the domain, which in immunoglobulin constant regions engages in intermolecular interactions.

Analysis of cDNA clones that code for the transmembrane forms of the mouse neural cell adhesion molecule (NCAM) and are generated by alternative RNA splicing.

The neural cell adhesion molecule (NCAM) exists in at least three different isoforms. In the mouse, NCAM proteins with apparent Mr's of 180,000, 140,000 and 120,000 have been distinguished. These are encoded by 4 to 5 different transcripts. Here we report the full amino acid sequence of an isoform which most likely represents NCAM-140. The N-terminal extracellular portion of the 829-residue polypeptide appears to be identical to all three NCAM proteins. The Mr of 91,276 is considerably smaller than the estimate based on SDS-gel electrophoresis. The 147 C-terminal residues are distinct from NCAM-120 and contain the putative transmembrane and cytoplasmic domains. The transcript encoding NCAM-140 contains almost 3.2 kb non-coding sequence with a canonical polyadenylation signal. While the 5' sequences of NCAM-140 hybridize with all NCAM mRNAs, the 3' probes recognize only the two larger transcripts of 7.4 and 6.7 kb. From S1 nuclease protection analyses and hybridization studies of several NCAM cDNA clones with genomic NCAM sequences one can conclude that the different NCAM transcripts are generated by alternative splicing. In addition to the two alternative splice sites in the sequence encoding the extracellular domains, a third one can be predicted approximately 320 nt downstream of the start of the NCAM-140-specific sequence portion. This finding is in agreement with the existence of an extra exon in the chicken NCAM-180. Comparison between mouse and chicken NCAM amino acid sequences revealed the highest homology in the second and fifth Ig-like domains and in the cytoplasmic parts suggesting that these regions serve highly conserved functions.

Identification of a cDNA clone that contains the complete coding sequence for a 140-kD rat NCAM polypeptide.

Neural cell adhesion molecules (NCAMs) are cell surface glycoproteins that appear to mediate cell-cell adhesion. In vertebrates NCAMs exist in at least three different polypeptide forms of apparent molecular masses 180, 140, and 120 kD. The 180- and 140-kD forms span the plasma membrane whereas the 120-kD form lacks a transmembrane region. In this study, we report the isolation of NCAM clones from an adult rat brain cDNA library. Sequence analysis indicated that the longest isolate, pR18, contains a 2,574 nucleotide open reading frame flanked by 208 bases of 5' and 409 bases of 3' untranslated sequence. The predicted polypeptide encoded by clone pR18 contains a single membrane-spanning region and a small cytoplasmic domain (120 amino acids), suggesting that it codes for a full-length 140-kD NCAM form. In Northern analysis, probes derived from 5' sequences of pR18, which presumably code for extracellular portions of the molecule hybridized to five discrete mRNA size classes (7.4, 6.7, 5.2, 4.3, and 2.9 kb) in adult rat brain but not to liver or muscle RNA. However, the 5.2- and 2.9-kb mRNA size classes did not hybridize to either a large restriction fragment or three oligonucleotides derived from the putative transmembrane coding region and regions that lie 3' to it. The 3' probes did hybridize to the 7.4-, 6.7-, and 4.3-kb message size classes. These combined results indicate that clone pR18 is derived from either the 7.4-, 6.7-, or 4.3-kb adult rat brain RNA size class. Comparison with chicken and mouse NCAM cDNA sequences suggests that pR18 represents the amino acid coding region of the 6.7- or 4.3-kb mRNA. The isolation of pR18, the first cDNA that contains the complete coding sequence of an NCAM polypeptide, unambiguously demonstrates the predicted linear amino acid sequence of this probable rat 140-kD polypeptide. This cDNA also contains a 30-base pair segment not found in NCAM cDNAs isolated from other species. The significance of this segment and other structural features of the 140-kD form of NCAM can now be studied.

Isolation and nucleotide sequence of mouse NCAM cDNA that codes for a Mr 79,000 polypeptide without a membrane-spanning region.

The neural cell adhesion molecule (NCAM) exists in several isoforms which are selectively expressed by different cell types and at different stages of development. In the mouse, three proteins with apparent Mr's of 180,000, 140,000 and 120,000 have been distinguished that are encoded by 4-5 different mRNAs. Here we report the full amino acid sequence of a NCAM protein inferred from the sequences of overlapping cDNA clones. The 706-residue polypeptide contains, towards its N-terminus, 5 domains that share structural homology with members of the immunoglobulin supergene family. The sequence does not encode a typical membrane-spanning segment, but ends with 24 uncharged amino acids followed by two stop codons. This fact, together with size considerations, make it highly likely that our sequence represents NCAM-120, which lacks transmembrane or cytoplasmic domains and is attached to the membrane by phospholipid. Probes from the 5' region detect all four NCAM gene transcripts present in mouse brain consistent with the notion that the extracellular domains are common to most NCAM forms. However, a 3' probe corresponding to the hydrophobic tail and non-coding region hybridizes specifically with the smallest mRNA species. S1 nuclease protection experiments indicate that this region is encoded by exon(s) spliced out from the other mRNAs. Furthermore, our clones that are highly homologous to a published chicken NCAM sequence which codes for putative transmembrane and cytoplasmic domains elsewhere, diverge from it at the presumptive splice junction. It appears thus that alternate use of exons determines whether NCAM proteins with membrane-spanning domains are synthesized.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of the human NCAM gene to band q23 of chromosome 11: the third gene coding for a cell interaction molecule mapped to the distal portion of the long arm of chromosome 11.

cDNA clones containing sequences coding for the murine neural cell adhesion molecule (N-CAM) were used in Southern hybridizations on human genomic DNA and demonstrated approximately 90% homology between human and murine NCAM genes. In situ hybridization with one of these clones was performed on human metaphase chromosomes and allowed the localization of the human NCAM gene to band q23 of chromosome 11. The genes for two other cell surface molecules believed to be involved in cell-cell interactions, Thy-1 and the delta chain of the T3-T cell receptor complex, have recently been localized to the same region of chromosome 11 in man. Moreover, this region of the human chromosome 11 appears to be syntenic to a region of murine chromosome 9 that also contains the staggerer locus: staggerer mice show abnormal neurological features which may be related to abnormalities in the conversion of the embryonic to the adult forms of the N-CAM molecule.

Transfection of murine LMTK- cells with purified HLA class I genes. VII. Association of allele- and locus-specific serological reactivities with respectively the first and second domains of the HLA-B7 molecule.

The individual contributions of the first two external domains of the HLA-B7 heavy chain to the expression of allele-specific (B7) and locus-specific (B and C) antigenic determinants were investigated using hybrid class I genes. Hybrid genes were constructed in vitro by exon shuffling between the parent genes HLA-B7, HLA-Cw3, HLA-A3, and H-2Kd, and their expression was monitored following transfection into mouse L cells. The results show that most allele-specific antigenic determinants are associated with the first external domain of the B7 heavy chain, whereas all the locus-specific antigenic determinants tested map to the second external domain.

Isolation of mouse N-CAM-related cDNA: detection and cloning using monoclonal antibodies.

Clones coding for the mouse neural cell adhesion molecule (N-CAM) were isolated from a cDNA library prepared in the expression vector lambda gt 11 from mRNA extracted from a mouse neuroblastoma cell line. This library was screened with two anti-N-CAM monoclonal antibodies directed against different sites on the molecule and with rabbit anti-N-CAM serum. Two clones were identified with the first monoclonal antibody, three with the second one, none reacted with both. The relevance of these cDNA clones to N-CAM was confirmed by several observations. First, cDNA sequences detected with one monoclonal antibody cross-hybridized with those identified by the other antibody. Second, the different fusion proteins all bound the rabbit serum in addition to one monoclonal antibody. Finally, the probes hybridized to discrete mRNA species of sufficient lengths to code for the very large N-CAM polypeptides in RNA preparations from N-CAM-expressing, but not from N-CAM-negative cells. An additional mRNA species not seen in embryonic brain was expressed in adult mouse brain. Genomic blot experiments indicated that sequences corresponding to one of our probes are present only a few times in the mouse genome.

Structural and functional studies on N-CAM neural cell adhesion molecules.

The neural cell adhesion molecules N-CAM are to date the best characterized adhesion molecules of the nervous system. They have a high content of sialic acid residues which are present in the form of unusual sialic acid polymers. During development, a 3 fold decrease in the sialic acid content is observed. These changes in the degree of sialylation profoundly affect the binding properties of the molecules. A subpopulation of mouse brain N-CAM bears a carbohydrate determinant shared with other brain cell surface proteins and with the HNK-1 antigen of natural killer cells. Not only the carbohydrate side chains but also the protein moieties of the N-CAMs are heterogeneous. Three polypeptides of 180 K, 140 K and 120 K have been characterized in mouse brain. The 180 K and 140 K chains span the membrane. They differ mainly by the length of their cytoplasmic extensions. These intracellular domains are unusually long and contain phosphorylated serine residues. The 120 K chain exists in two forms, one membrane-bound and one soluble. Earlier studies had shown the presence of N-CAM on neurones and astrocytes of the mouse central nervous system, whereas cultured astrocytes had been reported to be N-CAM-negative. Recent results show that N-CAM is also expressed on astrocytes in culture. To study expression and heterogeneity of N-CAM polypeptides at the mRNA and gene level, cDNA clones for mouse N-CAM have been isolated. They reveal multiple mRNA species in mouse brain. By contrast, the corresponding sequences seem to be present only a few times, perhaps only once, in the mouse genome.(ABSTRACT TRUNCATED AT 250 WORDS)

Allelism in the HLA class I multigene family.

Recent data on the structure of functional HLA class I genes shows that, at least at the HLA-A locus, the allelic genes are more related to each other than to HLA genes from other loci. This "A-ness" is discernible at the protein sequence level but much more evident when nucleotide sequences are compared; the homology is particularly striking in the 3' non-coding region. Genes coding for the same HLA specificity in different genetic backgrounds show no obvious difference, although in one case the 3' flanking regions are clearly different; the restriction maps around the HLA-A3 and HLA-AW24 genes are also compared to see whether the chromosomal environments of these two genes are recognizably similar.