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Electron microscopic mapping of RNA transcribed from the late region of polyoma virus DNA
Abstract:
The polyoma virus (Py) RNA species transcribed from the L DNA strand of the "late" region of the Py genome in Py-infected mouse cells have been mapped by hybridization with specific fragments of Py DNA followed by electron microscopic visualization of the hybrids. Total cellular polyadenylated Py-specific RNA molecules having an S value in the range of 16S to 20S were purified by oligodeoxythymidylic acidcellulose column chromatography, preparative hybridization with Py DNA, and sucrose gradient centrifugation. Cytoplasmic Py-specific RNA was similarily purified, except that it was not fractionated by sucrose gradient centrifugation. Hybrids of these RNA molecules and Py DNA fragments were spread for electron microscopy by either the cytochrome c technique or the bacteriophage T4 gene 32 protein method. The polyadenylic acid at the 3'-end of the RNA in the hybrids was identified by labeling with simian virus 40 DNA circles to which polybromodeoxyuridylic acid tails had been covalently attached. These experiments revealed the presence of three L DNA strand transcripts in both RNA preparations. Two of these RNA molecules were found to be spliced from chains transcribed from two noncontiguous parts of the late region. The third molecule either is a continuous transcript of the entire late region or contains a splicing feature which is too small to be reliably observed by the electron microscope methods used. The 5'-ends of the three RNA species map within a region extending from 68 to 70 map units on the Py restriction endonuclease map. Each of the two spliced molecules contains a 5'-terminal leader sequence transcribed from a DNA segment with an estimated length of 60 to 110 nuvleotides. The 3'-ends of the leaders map at 66.7 +/- 1.0 and 66.4 +/- 0.50 map units. In these molecules the 5'-ends of the other part (the main body) map at 59.4 +/- 0.90 and 49.4 +/- 2.0 map units, respectively. The 3'-termini of all three RNA species map at 24 to 25 map units.
Insights
Polyoma virus (Py) late region RNA transcripts were mapped using DNA hybridization and electron microscopy. Researchers identified three distinct RNA species, two of which are spliced, providing insights into viral gene expression.
Area of Science:
- Molecular Biology
- Virology
- Transcriptomics
Background:
- Polyoma virus (Py) plays a crucial role in cellular transformation and oncogenesis.
- Understanding the transcription of Py's late region is essential for deciphering its replication and pathogenic mechanisms.
- Previous studies have indicated complex RNA processing within the Py late region.
Purpose of the Study:
- To precisely map the RNA species transcribed from the late region of the Py genome.
- To identify and characterize spliced versus unspliced transcripts originating from the late region.
- To determine the precise locations of the 5' and 3' termini of these Py RNA transcripts.
Main Methods:
- Hybridization of Py RNA with specific Py DNA fragments.
- Electron microscopic visualization of RNA-DNA hybrids.
- Purification of polyadenylated and cytoplasmic Py-specific RNA using chromatography and centrifugation.
- Identification of polyadenylic acid tails using labeled SV40 DNA circles.
Main Results:
- Three distinct L DNA strand transcripts were identified in both total and cytoplasmic Py RNA.
- Two of these RNA molecules are confirmed to be spliced, originating from noncontiguous DNA segments.
- The 5' ends of the transcripts map between 68-70 map units, with leader sequences mapping at specific sites.
- The 3' termini of all three RNA species consistently map between 24-25 map units.
Conclusions:
- The late region of the Py genome produces at least three distinct RNA transcripts.
- Splicing is a key mechanism in generating diversity among Py late region RNAs.
- The defined mapping of these transcripts provides a foundational understanding for further functional studies of Py gene expression.