Report of a hitherto unreported sequence present and expressed in a cancer cell line
Introduction
During polymerase chain reaction (PCR)-based amplification of a 202 bp fragment of the N-ras gene around the 61 st codon, an additional fragment was routinely observed when DNA from a human cancer cell line, Hep2, was used as template. Single cell cultures were generated from the cell line and when DNA prepared from these subcultures was used as template in PCR reactions, the extra band always showed up, though it was absent when DNA extracted from human tissue was used as template. Cancer cell lines harbour many mutations, translocations and amplifications and such changes in this cell line have probably created extra sites of homology for the primers being used. As these changes often take place in genes which are relevant to the process of carcinogenesis, we decided to investigate the amplified region more closely. The sequence of this fragment has not been reported before. RT-PCR experiments indicate that part of this sequence is expressed at about 8 h after serum induction of Hep2 cells.
Section snippets
Cell culture
Human epidermoid carcinoma cell line Hep2 was cultured in DMEM supplemented with 2% fetal calf serum and antibiotics (100 IU/ml penicillin, 100 μg/ml streptomycin and fungizone).
DNA isolation
DNA was isolated by digestion of cells suspended in Tris (10 mM), EDTA (5 mM) and NaCl (150 mM) with proteinase K (50 μg/ml) and SDS (0.5%) at 50°C for 4 h. The digests were extracted with equal volumes of buffer-saturated phenol several times followed by phenol–chloroform and chloroform. DNA was precipitated first with
Results and discussion
Fig. 1 shows the amplification product after PCR of genomic DNA from two different sources using primers A and B. When the control human blood DNA was used as template, the expected band of 202 bp was obtained. However, when the Hep2 DNA was used as template, an extra band (X) of about 150 bp in length was obtained. When these PCR products were hybridized with the [α-35S]dATP-labelled 202 bp fragment, X failed to light up indicating that it was not a truncated version of the 202 bp band (data
Acknowledgements
Financial assistance from ICMR is acknowledged. N.D. is a CSIR senior research fellow.
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