Influence of altered gravity on intrachromosomal homologous recombination in mammalian cells

Abstract

We isolated mouse Ltk- cells stably transfected with a DNA recombination substrate containing two defective herpes simplex virus thymidine kinase (tk) genes. Cells in which an intrachromosomal recombination event occured between the tk genes were recovered by selecting for tk+ clones. Cells were cultured either under standard 1g conditions or cultured for 4 days under conditions of hypergravity (4g or 10g) at the NASA Ames Research Center (ARC) (Palo Alto, CA) prior to fluctuation analysis. To do these experiments, cells were shipped back and forth between our lab in South Carolina and ARC. Two groups of 1g control cells were used. One group was shipped to ARC and grown at 1g in the same NASA facility housing the equipment used for the hypergravity cultures, while a second group of cells was maintained at 1g in our lab in South Carolina. Our results obtained with two cell lines indicate that growth of cells at 4g or 10g has no significant effect on recombination. The rate of recombination for cells maintained under all conditions was approximately 1 x 10 −6 (events per generation), with cells shipped back and forth displaying perhaps slightly elevated rates compared with cells that remained in South Carolina. Recombination events were classified as either gene conversions or popouts (crossovers). We observed no significant differences in the spectrum of events recovered from cells grown under all conditions. Preliminary experiments using a clinostat to simulate microgravity reveal no significant effect of microgravity on recombination rate. We will further investigate the influence of microgravity on the nature of chromosomal recombination events as well as the influence of altered gravity on double-strand break-induced recombination in mammalian cells. This work was supported by NASA grant NCC 2-1374.