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Related Experiment Videos

Complete human rDNA repeat units isolated in yeast artificial chromosomes.

T Labella1, D Schlessinger

  • 1Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110.

Genomics
|November 1, 1989
PubMed
Summary

Researchers successfully isolated intact human ribosomal DNA (rDNA) repeat units using yeast artificial chromosomes (YACs). This breakthrough enables detailed study of rDNA structure and expression, revealing unexpected non-rDNA sequences in some clones.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Cell Biology

Background:

  • Human ribosomal DNA (rDNA) organization into large tandem repeats (44 kb) is known, with only a portion (13 kb) transcribed.
  • Conventional cloning systems struggle to accommodate the large size of intact rDNA repeat units, hindering direct structural analysis.

Purpose of the Study:

  • To isolate and characterize intact human ribosomal DNA repeat units.
  • To overcome limitations of conventional cloning systems for studying large genomic structures.
  • To provide a foundation for analyzing the expression of single rDNA units.

Main Methods:

  • Utilized yeast artificial chromosomes (YACs) as a cloning tool for large DNA fragments.
  • Employed a specific human rDNA spacer sequence as a probe for identifying YAC clones.

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  • Analyzed YAC inserts using restriction enzyme digestion (EcoRI, HindIII) and size determination.

    • Main Results:

    • Successfully isolated 13 YAC clones containing most or all of the rDNA repeat unit from 17,000 screened.
    • Eight clones were further analyzed, revealing 1 to 1.5 rDNA repeat units with expected restriction fragments.
    • Four larger clones (up to 950 kb) contained additional non-rDNA sequences flanking the rDNA repeats, suggesting genomic complexity.

    Conclusions:

    • Yeast artificial chromosomes are effective for cloning and analyzing large, intact human rDNA repeat units.
    • The presence of non-rDNA sequences in some clones indicates a more complex organization of nucleolar organizer regions than previously understood.
    • These findings offer a crucial resource for future research into rDNA structure, regulation, and expression.