Evidence for intrachromosomal gene conversion in cultured mouse cells

Area of Science:

• Molecular Biology
• Genetics
• Cell Biology

Background:

• Homologous recombination is a fundamental genetic process.
• Studying recombination between closely linked genes is challenging.
• Mammalian cell culture offers a model for genetic studies.

Purpose of the Study:

• To develop an experimental scheme for studying homologous recombination between closely linked genes.
• To quantify recombination frequencies in mammalian cells.
• To analyze the nature of recombination events.

Main Methods:

• Introduction of two distinct Xho I linker insertion mutants of the herpes simplex virus type 1 thymidine kinase (HTK) gene into mouse LTK- cells as direct repeats.
• Selection for TK+ (thymidine kinase positive) phenotype to identify recombinational events.
• Physical analysis of TK+ segregants using Xho I endonuclease digestion.

Main Results:

• TK+ segregants were observed at frequencies of 10(-4)-10(-5) in cell lines with both mutant genes.
• Control lines with a single mutant HTK gene showed significantly lower TK+ frequencies (≤10(-7)).
• Physical analysis confirmed the presence of a normal HTK gene in TK+ segregants, resistant to Xho I digestion.

Conclusions:

• The experimental scheme effectively facilitates the study of homologous recombination between closely linked genes.
• Recombination events occur at a measurable frequency in cultured mammalian cells.
• At least 50% of observed recombinants result from nonreciprocal exchanges (gene conversion).