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

Abstract

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.

Characterization of rat brain NCAM mRNA using DNA oligonucleotide probes

“…It did not recognize any mRNAs in adult rat liver which was run concomitantly in all experiments. When hybridizing the E7 probe to brain mRNA isolated from different stages of development, the same developmental changes in NCAM mRNAs as described in [6,19] were seen (data not shown). The 4.3 kb mRNA class was only present in small amounts and usually observed in rat brain postnatal days 4 and 10, where the total NCAM mRNA amount was highest.…”

“…From the same sequence an oligonucleotide specific for exon 17 followed by exon 19 (E17/19 probe, covering nucleotides 2648-2677) was constructed. Oligonucleotides specific for NCAM mRNA that either possess or lack the Pi exon were also constructed (EPi probe, nucleotides 1271-1300 and E7/8 probe, nucleotides 1257-1270 + 1301-1315) using the sequence given in [6]. Oligonucleotides were constructed from the sequence described in [11] Oligonucleotides were synthesized on a Biosearch 8750 DNAsynthesizer and labelled with 132P]dATP (NEN) using a DNA tailing kit from Boehringer Mannheim.…”

“…There are five different NCAM mRNA size classes of 7.4, 6.7, 5.2, 4.3 and 2.9 kb in adult rat brain [6]. More than 20 different exons are described that code for the distinct NCAM polypeptides [5,[7][8][9][10][11].…”

Identification of an Amino Acid Sequence Motif in the Cytoplasmic Domain of the NCAM‐140 kDa Isoform Essential for Its Neuritogenic Activity

“…Expression constructions coding for cytNCAM-180 and cyt-NCAM-140 were cloned into the modified vector pH␤Apr-1-neo by PCR using the rat NCAM-180 and rat NCAM-140 cDNAs (Small et al, 1987; GenBank accession number for NCAM-140, X06564) using the sense primer 5Ј-TAAGTC-GACCATGGTAAACAAGTGTGGCCTGCT-3Ј (incorporating the underlined SalI site, the Kozak sequence in bold, and TA nucleotides to preserve an open reading frame in italic) and the antisense primer 5Ј-AAATCTAGATCATGCTTTGCTCT-CATT-3Ј (incorporating the underlined XbaI site). The Nterminal and C-terminal truncated forms of the cytNCAM-140 were generated by PCR using the primer sets shown in Tables 1 and 2, respectively.…”

Characterization of cDNA clones encoding two putative isoforms of the α1 subunit of the dihydropyridine‐sensitive voltage‐dependent calcium channel isolated from rat brain and rat aorta

“…106 recombinants were screened with rabbit skeletal muscle clone ASKmCaChoel.3 [5]. The same probe was used to screen 50000 colonies of a rat brain cDNA library as in [14]. The positive clones were identified and subcloned into M13 mp18/19 by conventional techniques and DNA sequencing was performed using the dideoxy chain termination method [5].…”