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Related Experiment Videos

Human complement factor B: cDNA cloning, nucleotide sequencing, phenotypic conversion by site-directed mutagenesis

T Horiuchi1, S Kim, M Matsumoto

  • 1First Department of Internal Medicine, School of Medicine, Ehime University, Japan.

Molecular Immunology
|December 1, 1993
PubMed
Summary

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Researchers isolated and sequenced the human factor B (fB) cDNA clone BHL4-1. They found that wild-type and mutant fB alleles, including a Q7R mutant, exhibit similar hemolytic activity, suggesting structural variations do not impact function.

Area of Science:

  • Molecular Biology
  • Immunology
  • Biochemistry

Background:

  • Factor B (fB) is a crucial component of the alternative complement pathway.
  • Understanding fB gene structure and function is vital for immunological research.

Purpose of the Study:

  • To isolate and characterize the full-length cDNA clone of human factor B.
  • To investigate the functional impact of specific genetic variations on factor B activity.

Main Methods:

  • Isolation of a full-length cDNA clone (BHL4-1) from a human liver cDNA library.
  • Sequencing of the cDNA to determine its structure.
  • Site-directed mutagenesis to create a Q7R mutant.
  • Transient expression of wild-type and mutant factor B in a eukaryotic system.

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  • Assay of specific hemolytic activities.

    • Main Results:

    • The BHL4-1 clone comprises 2388 bp, including untranslated regions and a 2292 bp open reading frame.
    • The deduced amino acid sequence corresponds to the F allele of factor B, with a leader peptide and mature polypeptide chain.
    • No significant difference in specific hemolytic activity was observed between wild-type factor B, a Q7R mutant, and native factor B.

    Conclusions:

    • The characterized fB cDNA clone provides a basis for further functional studies.
    • The specific amino acid substitution (Q7R) in factor B does not significantly alter its hemolytic activity.
    • Structural variations may not be the sole determinants of factor B function in the complement system.