The Chinese hamster HPRT gene: restriction map, sequence analysis, and multiplex PCR deletion screen

Area of Science:

• Genetics
• Molecular Biology
• Genomics

Background:

• The Chinese hamster hypoxanthine guanine phosphoribosyltransferase (HPRT) gene is a crucial target for in vitro mutation studies due to facile mutant selection.
• Understanding the HPRT gene's fine structure is essential for investigating molecular defects and developing accurate mutation detection methods.

Purpose of the Study:

• To determine the complete fine structure of the Chinese hamster HPRT gene, including exon-intron boundaries and flanking sequences.
• To develop a rapid and specific method for identifying deletions within the HPRT gene in mutant cell lines.

Main Methods:

• Gene sequencing of all nine exons and flanking regions using lambda bacteriophage cloning and inverse polymerase chain reaction (PCR).
• Comparative sequence analysis of the Chinese hamster HPRT gene with mouse and human orthologs.
• Development and application of a multiplex PCR assay using primers derived from flanking sequences to amplify exon-containing fragments.

Main Results:

• The Chinese hamster HPRT gene comprises nine exons spanning 36 kb of DNA.
• Sequence homology was observed in flanking regions of exons 2-9 across species, with less conservation around exon 1.
• A multiplex PCR assay successfully identified total and partial HPRT gene deletions in Chinese hamster mutants.

Conclusions:

• The detailed characterization of the Chinese hamster HPRT gene provides a foundation for molecular defect analysis.
• The developed multiplex PCR assay offers a faster and more specific alternative to Southern blot analysis for detecting HPRT gene deletions.
• This research enhances the tools available for studying mutations in the HPRT gene, a widely used model system.