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Author(s): J. R. Ledwell (corresponding author); E. T. Montgomery; K. L. Polzin; L. C. St. Laurent; R. W. Schmitt; J. M. Toole The overturning circulation of the ocean plays an [...]

Aptamers are double-stranded DNA or single-stranded RNA molecules that bind specific molecular targets. Large randomly generated populations can be enriched in aptamers by in vitro selection and polymerase chain reaction. But so far single-stranded DNA has not been investigated for aptamer properties, nor has a target protein been considered that does not interact physiologically with nucleic acid. Here we describe the isolation of single-stranded DNA aptamers to the protease thrombin of the blood coagulation cascade and report binding affinities in the range 25-200 nM. Sequence data from 32 thrombin aptamers, selected from a pool of DNA containing 60 nucleotides of random sequence, displayed a highly conserved 14-17-base region. Several of these aptamers at nanomolar concentrations inhibited thrombin-catalysed fibrin-clot formation in vitro using either purified fibrinogen or human plasma.

A complete copy of the mRNA sequences encoding human coagulation factor VIII:C has been cloned and expressed. The DNA sequence predicts a single chain precursor of 2,351 amino acids with a relative molecular mass (Mr) 267,039. The protein has an obvious domain structure, contains sequence repeats and is structurally related to factor V and ceruloplasmin.

DNA sequences capable of inducing oncogenic transformation of NIH3T3 mouse cells are found in a number of human tumour cell lines. When DNAs of these cell lines are applied to monolayer cultures of the mouse fibroblasts, foci of transformed cells are observed 2-3 weeks later. DNA from cells of such primary foci can be used in turn to induce foci in a second cycle of gene transfer. The human DNA sequences responsible for transformation have been called oncogenes, the best characterized of which is closely related to the Harvey murine sarcoma virus oncogene. Here we present a characterization of an oncogene which we found originally to be present in DNA of the SW480 colon carcinoma cell line. We indicate its structural outlines and demonstrate, in extension of reported results, its presence in an activated form in the genome of several types of human tumour cell lines as well as in biopsy tissue from an adenocarcinoma of the large bowel. We identify this tumour oncogene with c-Ki-ras2, one of two known members of the Kirsten ras family of human proto-oncogenes, extending a series of recent reports which have demonstrated homologies between human oncogenes and those of Harvey and Kirsten murine sarcoma viruses. The c-Ki-ras2 oncogene of several tumour cell lines is shown to be amplified.