Complementary DNA cloning of a receptor for tumor necrosis factor and demonstration of a shed form of the receptor

Journal Article · Wed Aug 01 04:00:00 EDT 1990 · Proceedings of the National Academy of Sciences of the United States of America; (USA)

DOI:https://doi.org/10.1073/pnas.87.16.6151· OSTI ID:5851451

Heller, R A; Song, Kyung; Onasch, M A; Chang, D; Ringold, G M ^[1]; Fischer, W H ^[2]

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1. Institute of Cancer and Developmental Biology, Palo Alto, CA (USA)
2. The Salk Institute, La Jolla, CA (USA)

Tumor necrosis factor (TNF) receptor (TNFR) was isolated as a 68-kDa glycoprotein from UC/HeLa 2-5 cells developed from a parental B-cell line (UC cells) to overexpress the receptor. Tryptic digests of two separate TNFR preparations provided amino acid sequences of four different peptides. Amino-terminal analysis indicated the presence of the amino-acid sequence Val-Ala-Phe-Thr-Pro, reported to be the amino-terminal sequence of a 30-kDa urinary TNF-binding protein II. Examination of the cultured medium of UC/HeLa 2-5 cells showed an abundance of a 40-kDa TNF-binding protein, indicating that the previously cited 30-kDa TNF-binding protein II is likely to be a shed form of the TNFR. Based on the peptide sequences, oligonucleotides were synthesized, and two of these were used as primers in the polymerase chain reaction to amplify cDNA sequences from poly(A){sup +} RNA of UC/HeLa 2-5 cells. These PCR fragments were radiolabeled and used to screen a cDNA library made from UC/HeLa 2-5 mRNA. Further analysis identified cDNA sequences that encoded the amino acid sequences of all four TNFR peptides. RNA blot-hybridization analysis of UC/HeLa 2-5 mRNA revealed a 3.8-kilobase transcript of the same size as the mRNA in the parental UC cells. Genomic Southern blots indicated the presence of a single gene in parental cells and a second, amplified gene in TNFR-overexpressing cells, suggesting amplification of the transfected gene as a possible mechanism for the increase in TNFR numbers in UC/HeLa 2-5 cells.

• OSTI ID: 5851451
• Journal Information: Proceedings of the National Academy of Sciences of the United States of America; (USA), Journal Name: Proceedings of the National Academy of Sciences of the United States of America; (USA) Vol. 87:16; ISSN 0027-8424; ISSN PNASA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMINO ACID SEQUENCE
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CLONING
DAYS LIVING RADIOISOTOPES
DISEASES
DNA HYBRIDIZATION
DNA-CLONING
ELECTRON CAPTURE RADIOISOTOPES
ELECTROPHORESIS
EVEN-ODD NUCLEI
GENE AMPLIFICATION
HELA CELLS
HYBRIDIZATION
INFLAMMATION
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ISOTOPES
LIGHT NUCLEI
MEMBRANE PROTEINS
MESSENGER-RNA
MOLECULAR STRUCTURE
NECROSIS
NEOPLASMS
NUCLEI
NUCLEIC ACIDS
ODD-EVEN NUCLEI
ODD-ODD NUCLEI
OLIGONUCLEOTIDES
ORGANIC COMPOUNDS
PATHOLOGICAL CHANGES
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PROTEINS
PURIFICATION
RADIOISOTOPES
RECEPTORS
RNA
SULFUR 35
SULFUR ISOTOPES
SYMPTOMS