Amplification of a gene coding for human T-cell differentiation antigen

Area of Science:

• Immunology
• Molecular Biology
• Genetics

Background:

• The human T-cell differentiation antigen Leu-2 (also known as T8) is a marker for T lymphocytes with cytotoxic or suppressor functions.
• Gene amplification is a process where a cell increases the number of copies of a specific gene, often leading to altered protein expression.

Purpose of the Study:

• To investigate spontaneous gene amplification in mouse L-cells transfected with the human Leu-2 antigen.
• To characterize the changes in Leu-2 expression and DNA in cells exhibiting amplified gene sequences.

Main Methods:

• Mouse L-cells were transfected with the gene for the human T-cell antigen Leu-2.
• Cells expressing high levels of Leu-2 were isolated using fluorescence-activated cell sorting (FACS) after repeated rounds of selection and regrowth.
• DNA analysis was performed to assess gene copy number and identify chromosomal abnormalities.

Main Results:

• A population of cells with a 40-fold increase in Leu-2 antigen staining intensity was generated.
• These cells showed a 20-fold increase in DNA transformation efficiency for Leu-2.
• Significant amplification of human DNA sequences (10- to 50-fold) and the presence of double minute chromosomes were observed.

Conclusions:

• This study provides the first evidence of spontaneous gene amplification for membrane antigens in cultured cells.
• The findings suggest that selective pressure can drive gene amplification, leading to significantly enhanced antigen expression.
• The observed double minute chromosomes are indicative of a mechanism of gene amplification common in mammalian cells.