Cloning and characterization of an alternative splicing transcript of the gene coding for human cytidine deaminase

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Human cytidine deaminase (HCD) is crucial for nucleoside metabolism.
• The HCD gene structure suggests potential for alternative splicing, though no variants were previously reported.

Purpose of the Study:

• To identify and characterize a potential alternative splicing product of the HCD gene.
• To investigate the functional consequences of this novel variant.

Main Methods:

• DNA sequence alignment to identify structural differences.
• Gene expression in E. coli with a GST tag for protein production.
• Western blot and mass spectrometry for molecular mass confirmation.
• Enzyme activity assays and cell-based assays using NIH3T3 cells.

Main Results:

• A smaller HCD variant, termed HSCD, was cloned and characterized.
• HSCD results from alternative splicing, joining exons 1 and 4 and deleting exons 2 and 3, causing a 170 bp deletion.
• This splicing event induces a reading frame shift, leading to a predicted 9.8 kDa protein.
• HSCD demonstrated a complete loss of cytidine deaminase activity.
• NIH3T3 cells expressing HSCD showed resistance to cytosine arabinoside, confirming loss of function.

Conclusions:

• HSCD is a novel, alternatively spliced product of the HCD gene.
• The structural alterations in HSCD result in a non-functional enzyme.
• This finding expands our understanding of HCD gene regulation and its functional implications.