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The functional human dihydrofolate reductase gene.

M J Chen, T Shimada, A D Moulton

    The Journal of Biological Chemistry
    |March 25, 1984
    PubMed
    Summary
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    Researchers characterized the human dihydrofolate reductase (DHFR) gene, detailing its structure, promoter region, and transcription start site. This analysis clarifies the origins of different DHFR mRNA species in human cells.

    Area of Science:

    • Molecular Biology
    • Genetics
    • Genomics

    Background:

    • The dihydrofolate reductase (DHFR) gene is crucial for cellular metabolism and is a target for various therapeutic agents.
    • Understanding the precise structure and regulatory elements of the human DHFR gene is essential for comprehending gene expression and potential dysregulation.

    Purpose of the Study:

    • To molecularly clone, map, and DNA sequence the functional human dihydrofolate reductase (DHFR) gene.
    • To identify the DHFR gene promoter and transcription initiation site.
    • To compare the human DHFR gene with its mouse counterpart to understand evolutionary conservation and differences.

    Main Methods:

    • Utilized molecular cloning, DNA mapping, and DNA sequencing techniques.
    • Constructed a DHFR minigene in a plasmid expression vector.

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  • Performed in vitro transcription reactions and transfected monkey kidney and human HeLa cells to determine transcription initiation and localization.

    • Main Results:

    • The human DHFR gene is approximately 30 kilobases long, with coding sequences divided into 6 exons and 5 introns of varying lengths.
    • The DHFR gene promoter was identified upstream of the initiation codon, with transcription initiating 71 +/- 2 base pairs upstream.
    • Sequence homology between human and mouse DHFR genes is primarily in coding regions and the 3' untranslated region, with differences in the 5' flanking region.

    Conclusions:

    • The identified transcription start site and known polyadenylation sites explain the different DHFR mRNA species observed in human cells.
    • Comparative analysis reveals conserved and divergent features between human and mouse DHFR genes, offering insights into gene regulation and evolution.
    • Detailed characterization of the human DHFR gene provides a foundation for further studies on gene function, regulation, and disease association.