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PCR walking from microdissection clone M54 identifies three exons from the human gene for the neural cell adhesion molecule L1 (CAM-L1).

ABSTRACT: Microdissection has proved to be a powerful tool in the construction of libraries from specific chromosome segments (11) which are poorly covered by existing RFLP markers. Microclones also represent starting points for finding genes of interest. However, their length (100 to 200 bp) can make their use as probes problematic and identifying them as coding sequence is difficult. We report here that microclones can be extended in vitro by a modified version of our original PCR walking method (10) which utilises oligo-cassettes and the solid phase biotin/streptavidin separation system. We have extended the microclone M54, derived by dissection from Xq27.2 to proximal Xq28 (12), in both directions for approximately 700 bp. Direct sequencing of these products revealed that M54 was located within an intron of the human gene encoding the neural cell adhesion molecule L1 (CAM-L1) which has been recently mapped to Xq28 (13). The extension of M54 also identified three exons of this gene. This information allowed subsequent amplification of a 2.4 kb cDNA molecule from fetal human brain mRNA which encodes most of human CAM-L1. Sequencing of this cDNA revealed a high degree of sequence conservation with the mouse homologue (14). This is the first description of extension of a human derived microclone by PCR mediated walking within total human genomic DNA. These results show that anonymous DNA sequences may be extended into coding or any sequence.

SUBMITTER: Rosenthal A

PROVIDER: S-EPMC328904 | biostudies-other | 1991 Oct

REPOSITORIES: biostudies-other

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PCR walking from microdissection clone M54 identifies three exons from the human gene for the neural cell adhesion molecule L1 (CAM-L1).

Rosenthal A A MacKinnon R N RN Jones D S DS

Nucleic acids research 19911001 19

Microdissection has proved to be a powerful tool in the construction of libraries from specific chromosome segments (11) which are poorly covered by existing RFLP markers. Microclones also represent starting points for finding genes of interest. However, their length (100 to 200 bp) can make their use as probes problematic and identifying them as coding sequence is difficult. We report here that microclones can be extended in vitro by a modified version of our original PCR walking method (10) wh ...[more]

PMID: 1923824

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Project description:Diverse cell-surface molecules of the nervous system play an important role in specifying cell interactions during development. Using a method designed to generate mAbs against neural surface molecules of defined molecular weight, we have previously reported on the surface protein, Bravo, found in the developing avian retinotectal system. Bravo is immunologically detected on developing optic fibers in the retina, but absent from distal regions of the same fibers in the tectum. We have isolated cDNA clones encompassing the entire coding region of Bravo, including clones containing five alternative sequences of cDNA. These putative alternatively spliced sequences encode stretches of polypeptide ranging in length from 10-93 amino acids and are predicted to be both extra- and intracellular. The deduced primary structure of Bravo reveals that, like the cell adhesion molecules (CAMs) chicken Ng-CAM and mouse L1, Bravo is composed of six Ig-like domains, five fibronectin type III repeats, a transmembrane domain, and a short cytoplasmic region. Recently, the cDNA sequence of a related molecule, Nr-CAM, was reported and its possible identity with Bravo discussed (Grumet, M., V. Mauro, M. P. Burgoon, G. E. Edelman, and B. A. Cunningham. 1991. J. Cell Biol. 113:1399-1412). Here we confirm this identity and moreover show that Bravo is found on Müller glial processes and end-feet in the developing retina. In contrast to the single polypeptide chain structure of Nr-CAM reported previously, we show that Bravo has a heterodimer structure composed of an alpha chain of M(r) 140/130 and a beta chain of 60-80 kD. As with L1 and Ng-CAM, the two chains of Bravo are generated from an intact polypeptide by cleavage at identical locations and conserved sites within all three molecules (Ser-Arg/Lys-Arg). The similar domain composition and heterodimer structure, as well as the 40% amino acid sequence identity of these molecules, defines them as an evolutionarily related subgroup of CAMs. The relationship of Bravo to molecules known to be involved in cell adhesion and process outgrowth, combined with its pattern of expression and numerous potential isoforms, suggests a complex role for this molecule in cell interactions during neural development.

Project description:Neuron-glia cell adhesion molecule (Ng-CAM) mediates cell adhesion between neurons homophilically and between neurons and glia heterophilically; it also promotes neurite outgrowth. In the chick brain, Ng-CAM is detected as glycoproteins of 190 and 210 kD (Ng-CAM200) with posttranslational cleavage products of 135 kD (F135, which contains most of the extracellular region) and 80 kD (F80, which includes the transmembrane and the cytoplasmic domains). To examine the functions of each of these components, we have expressed Ng-CAM200, F135, and F80 in murine L cells, and F135 and F80 as GST fusion proteins in the pGEX vector in bacteria. Appropriately transfected L cells expressed each of these proteins on their surfaces; F135 was also found in the media of cells transfected with Ng-CAM200 and F135. In addition to binding homophilically, cells transfected with Ng-CAM200 and F135 bound heterophilically to untransfected L cells, suggesting that there is a ligand for Ng-CAM on fibroblasts that may be related to the glial ligand. Detailed studies using the transfected cells and the fusion proteins indicated that both the homophilic and the heterophilic binding activities of Ng-CAM are localized in the F135 fragment of the molecule. The results also indicated that proteolytic cleavage of Ng-CAM200 is not required either for its expression on the cell surface or for cell adhesion and that there is an "anchor" for F135 on L cells (and presumably on neurons). In contrast to the cell binding results, the F80 but not the F135 fusion protein enhanced the outgrowth of neurites from dorsal root ganglion cells; this activity was associated with the FnIII repeats of F80. The observations that a protein corresponding to F135 contains the cell aggregation sites whereas one corresponding to the F80 has the ability to promote neurite outgrowth suggest that proteolytic cleavage may be an important event in regulating these Ng-CAM activities during embryonic development and neural regeneration.

Dataset Information

PCR walking from microdissection clone M54 identifies three exons from the human gene for the neural cell adhesion molecule L1 (CAM-L1).

Publications

PCR walking from microdissection clone M54 identifies three exons from the human gene for the neural cell adhesion molecule L1 (CAM-L1).

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