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Purification of tDNA from yeast
Hoppe-Seyler'S Zeitschrift Fur Physiologische Chemie
|November 1, 1975
Summary
Researchers isolated yeast transfer RNA-DNA (tRNA-tDNA) hybrids. These hybrids reveal that transfer RNA (tRNA) genes are arranged in tandem, with preserved spacer DNA, offering insights into gene organization.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Transfer RNA (tRNA) genes are essential for protein synthesis.
- Understanding the organization and structure of tRNA gene clusters is crucial for comprehending gene regulation and genome evolution.
- Previous studies have explored tRNA gene organization, but direct isolation and characterization of tRNA-DNA hybrids in yeast were limited.
Purpose of the Study:
- To isolate and characterize tRNA-tDNA hybrids from yeast.
- To investigate the arrangement of tRNA cistrons within the yeast genome.
- To determine if external spacer DNA is preserved in the isolated tDNA.
Main Methods:
- Isolation of tRNA-tDNA hybrids using BD-cellulose chromatography with salt gradients and formamide.
- Characterization of hybrids via density centrifugation in cesium sulfate (CS2SO4).
- Enzymatic treatment with alkali and pancreatic ribonuclease to confirm hybrid structure.
Main Results:
- Successful isolation of yeast tRNA-tDNA hybrids.
- Density centrifugation confirmed the hybrid nature of the isolated material.
- Alkali and ribonuclease treatments validated the presence of both RNA and DNA components.
- Experiments using sheared DNA indicated tandem arrangement of tRNA cistrons.
- Evidence suggests preservation of external spacer DNA in the tDNA component.
Conclusions:
- Yeast tRNA-tDNA hybrids can be effectively isolated and purified.
- The study provides strong evidence for tandem arrangement of tRNA cistrons in yeast.
- External spacer DNA sequences are likely maintained within the tDNA during hybrid formation, offering insights into gene clustering and processing.