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Related Experiment Videos

Rabbit Ig kappa 1b6 gene structure.

K L Dreher1, V Asundi, B Wolf

  • 1Weis Center for Research, Geisinger Clinic, Danville, PA 17822.

Journal of Immunology (Baltimore, Md. : 1950)
|July 1, 1990
PubMed
Summary
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Unexpected kappa-homologous sequences in rabbit DNA were identified as the kappa 1 gene. Molecular cloning revealed a glycine to serine change, creating an EcoRI site and confirming the kappa 1 gene identity.

Area of Science:

  • Immunogenetics
  • Molecular Biology
  • Genomics

Background:

  • Southern blot analyses previously detected unexpected kappa-homologous sequences in EcoRI-digested DNA from kappa 1b6 homozygous rabbits and RMH H158 cell lines.
  • These findings were surprising due to the incompatibility of the published kappa 1b6 C region protein sequence with an EcoRI restriction site at the nucleotide level.

Purpose of the Study:

  • To identify the nature of the unexpected kappa-homologous sequences detected in kappa 1b6 homozygous rabbits and RMH H158 cell lines.
  • To resolve the discrepancy between the published protein sequence and the presence of EcoRI sites in the kappa 1b6 gene locus.

Main Methods:

  • Molecular cloning of kappa-homologous sequences from DNA of the kappa 1b6 L chain secreting RMH H158 cell line.
  • Structural analyses of the cloned sequences to determine their genetic information.

Related Experiment Videos

  • Deduction of protein sequence from the kappa 1b6 C region gene and comparison with the published sequence.
  • Genomic Southern blot analyses to confirm structural assignments.

    • Main Results:

    • Molecular cloning and structural analysis revealed that the isolated sequences encode the majority of the kappa 1b6 L chain gene locus.
    • The deduced protein sequence from the kappa 1b6 C region gene differed from the published sequence at five amino acid positions.
    • One amino acid substitution (glycine to serine) introduced an EcoRI restriction endonuclease recognition site within the kappa 1b6 C region gene.
    • Genomic Southern blot analyses confirmed these findings, identifying the EcoRI-sensitive fragments as the kappa 1 gene.

    Conclusions:

    • The unexpected EcoRI-sensitive kappa-homologous fragments in RMH H158 cells and kappa 1b6 rabbits represent the nominal kappa 1 gene.
    • The discrepancy was due to an unpredicted amino acid substitution creating an EcoRI site, not an alternative isotype or pseudogene.
    • The isolated kappa 1b6 J-C gene locus shares common structural features with other identified kappa 1 alleles, supporting its classification.