Chloroquine treatment of rodent cells during the first hours of polyoma DNA transfection increase the fraction of cells expressing viral functions. The effect has been observed after DNA absorption using both the DEAE-dextran and calcium phosphate coprecipitation methods. Exposure to chloroquine increased the proportion of transfected mouse cells to approximately 40%. From a culture of one million such cells, microgram quantities of newly synthesized viral DNA could be isolated. Similarly, the transformation frequency of rat cells following polyoma DNA transfection was approximately 6-fold increased by chloroquine treatment. The effect of the compound was even more pronounced in transfections with linear forms of polyoma DNA, suggesting that chloroquine inhibits degradation of DNA absorbed by the cells.

High efficiency polyoma DNA transfection of chloroquine treated cells

https://www.pnas.org/content/pnas/48/12/2026.full.pdf

Genetics of human cell lines, iv. dna-mediated heritable transformation of a biochemical trait

Although it is accepted on the basis of biological and morphological evidence that mammalian cells will take up macromolecules, little is known about the kinetics, the specificity, and the functions of this uptake. With labeled proteins used as models, it is found that the transport proceeds at very low rates, requires little energy, and is markedly enhanced by polybasic compounds. Molecular charge and size are important factors: cells clearly favor cationic macromolecules of large molecular weights. Neither factor, however, can fully account for the selectivity detected in the uptake of different proteins. Ingested albumin undergoes rapid and extensive degradation. This fact suggests that macromolecules have only a limited chance to express their biological activity in target cells, a finding that is relevant also to the role of foreign nucleic acids and the possibility of achieving genetic transformation in animal cells. There are concrete indications, however, that in spite of their short half-life, proteins can act as carriers, as precursors of active agents, and as regulators of metabolic functions in host cells. They may also be important in the control of growth and differentiation. These functions of exogenous proteins are still largely unexplored.

Uptake of protein by mammalian cells: an underdeveloped area. The penetration of foreign proteins into mammalian cells can be measured and their functions explored.

Induction of a virus infection by cloned simian virus 40 DNA was chosen as a test system to detect transfer of genes from bacteria to cultured mammalian cells. Escherichia coli cells containing a recombinant plasmid with three tandem inserts of simian virus 40 DNA were able to infect CV-1 monkey cells under various conditions. The gene transfer was resistant to DNase I and therefore seems not to occur via free DNA but most likely via uptake of whole bacteria, followed by release of plasmid DNA and generation of infectious circular simian virus 40 DNA in a recombination-excision process. Spontaneous transfer was found to be infrequent, 4 x 10(9) bacteria yielding one infection per 10(7) monkey cells. The frequency was greatly increased by adding bacteria as a calcium phosphate coprecipitate or by fusion of lysozyme-treated bacteria (protoplasts) with monkey cells in the presence of polyethylene glycol. With the latter technique, 10(4) protoplasts gave rise to one infection per 15 monkey cells. Experiments with other cell lines of human, monkey, and mouse origin, and also with bacteria harboring another recombinant plasmid, indicate that DNA transfer from bacteria to mammalian cells is a general phenomenon.

https://www.pnas.org/content/pnas/77/4/2163.full.pdf

Direct transfer of cloned genes from bacteria to mammalian cells

Vaccinia virus which had its DNA labeled with thymidine-H3 was purified and used as inoculum for L cells growing in suspension. Samples taken over an 8-hour period after infection were studied by light and electron microscopic autoradiography. Within 20 minutes of its being taken up at the cell membrane in phagocytic vesicles, the outer coat of vaccinia becomes disrupted and the virus core containing the labeled DNA passes into the cytoplasmic matrix. Within 1 hour after inoculation the labeled material passes out of the cores into zones of viroplasm, where cores or remnants of cores are gathered and the label becomes more concentrated by 3 hours after inoculation. Most of the label is conserved in the viroplasm areas during the remainder of the experiment. However, 6 hours after inoculation a very small proportion of progeny virus in the cytoplasm, morphologically distinct from the cores of the inoculum, has associated with it labeled material, perhaps derived from the DNA of the inoculum.

https://rupress.org/jcb/article-pdf/18/1/51/1337043/51.pdf

A. Bendich

Papers

Isolation of infectious deoxyribonucleic acid from se polyoma-infected tissue cultures