A growth factor for animal cells derived from peptone : II. Effect of the growth factor on cellular metabolism

A growth factor for cultured mammalian cells was isolated from peptone by means of Sephadex, Dowex 50W and paper chromatography. It had a mol. wt of about 700 and was stable in both acidic or alkali conditions. It did not contain carbohydrates, nucleic acids or aromatic compounds but reacted strongly with ninhydrin. Amino acid analysis showed that glycine and serine constituted about 96% of the amino acids, but small amounts of lysine, aspartic acid, glutamic acid, threonine and alanine were also present.Minimum medium supplemented with the growth factor could support the growth of L-DC cells in vitro to the same extent as peptone or serum supplemented medium, and cells could be maintained continuously in growth factor supplemented medium.     

Characterization of an episome produced in hamster cells that amplify a transfected CAD gene at high frequency: functional evidence for a mammalian replication origin.

In a previous study (G. M. Wahl, B. Robert de Saint Vincent, and M. L. De Rose, Nature (London) 307:516-520, 1984), we used gene transfer of a CAD cosmid to demonstrate that gene position profoundly affects amplification frequency. One transformant, T5, amplified the donated CAD genes at a frequency at least 100-fold higher than did the other transformants analyzed. The CAD genes in T5 and two drug-resistant derivatives were chromosomally located. In this report, we show that a subclone of T5 gives rise to an extrachromosomal molecule (CAD episome) containing the donated CAD genes. Gel electrophoresis indicated that the CAD episome is approximately 250 to 300 kilobase pairs, and a variety of methods showed that it is a covalently closed circle. We show that the CAD episome replicates semiconservatively and approximately once per cell cycle. Since the CAD cosmid, which comprises most of the CAD episome, does not replicate autonomously when transfected into cells, our results indicate that either the process which generated the episome resulted in a cellular origin of DNA replication being linked to the CAD sequences or specific rearrangements within the episome generated a functional origin. The implications of these results for mechanisms of gene amplification and the genesis of minute chromosomes are discussed.