Cloning and expression of alternative transcripts of a novel neuroendocrine-specific gene and identification of its 135-kDa translational product.
Monoclonal antibodies RNL-2 and RNL-3 were previously shown to react with four 35-45-kDa proteins, expressed only in small cell lung carcinoma NCI-H82 cells, but to stain a subset of neuroendocrine tissues and neoplasms (Broers, J. L. V., Mijnheere, E. P., Klein Rot, M., Schaart, G., Sijlmans, A., Boerman, O. C., and Ramaekers, F. C. S. (1991) Cancer 67, 619-633). We used RNL-2 and RNL-3 to isolate cDNA sequences that code for proteins containing the two corresponding epitopes and utilized such cDNAs to develop second generation antibodies. Using these antibodies, we identified a novel 135-kDa protein. The corresponding cDNAs were found to belong to a previously unknown gene with a neuroendocrine-specific expression pattern, tentatively designated NSP gene. NSP transcription appeared to result in mRNAs of 3.4 and 1.8 kilobases (kb). In the NCI-H82 cells only, an apparently aberrant transcript of 2.3 kb was found. cDNAs containing coding sequences of the 3.4-, 2.3-, and 1.8-kb transcripts were isolated, and nucleotide sequence analysis revealed extensive sequence overlap and open reading frames for proteins of 776, 356, and 208 amino acids, respectively. The three deduced proteins all have a common carboxyl-terminal part with two large hydrophobic regions. Transfection of the complete coding sequences of the 3.4-kb transcript resulted in the production of a protein with an apparent molecular mass of 135 kDa. This protein is predicted to be highly negatively charged (calculated pI of 4.35), to be rich in proline and serine, and to contain multiple potential phosphorylation sites.
Roebroek
A J
AJ
Laboratory for Molecular Oncology, University of Leuven, Belgium.
van de Velde
H J
HJ
Van Bokhoven
A
A
Broers
J L
JL
Ramaekers
F C
FC
Van de Ven
W J
WJ
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D004247:DNA; D000939:Epitopes; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics*; D051381:Rats; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D000939:Epitopes; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; C081781:RTN1 protein, human; C489914:Rtn1 protein, mouse; C489913:Rtn1 protein, rat; D004247:DNA