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Transcription of intracisternal A-particle genes in mouse myeloma and Ltk- cells
Abstract:
In vivo- and in vitro-synthesized RNA from the murine plasmacytoma MOPC-315 was found to hybridize to all regions of a 7.2-kilobase-pair intracisternal A-particle (IAP) gene. IAP-specific transcripts were also detected in mouse Ltk- cells, but not in cells derived from normal tissues (kidney, liver, spleen) of 6-week-old BALB/c mice. Three RNA species of 7.2, 5.3, and 3.8 kilobases were identified by Northern blot analysis of MOPC-315 polyadenylated RNA. The 7.2- and 5.3-kilobase transcripts were found in greater levels in nuclear as compared with whole cell RNA, suggesting the involvement of one or more of the following mechanisms: RNA processing, preferential nuclear transport, or differential RNA stability. We show that the primary IAP transcript is initiated within the long terminal repeat by hybridization analysis with restriction digests of cloned IAP DNA and [gamma-S]pppApNp ... RNA synthesized in nuclei with [gamma-S]ATP as the RNA initiating probe. Low concentrations of alpha-amanitin (2 micrograms/ml) inhibited IAP RNA synthesis by greater than 90%, suggesting that RNA polymerase II is responsible for IAP transcription.
Insights
Intracisternal A-particle (IAP) gene transcripts were detected in mouse plasmacytoma cells but not normal tissues. RNA polymerase II transcribes the primary IAP transcript, initiated within the long terminal repeat.
Area of Science:
- Molecular Biology
- Genetics
- Virology
Background:
- Intracisternal A-particle (IAP) genes are retrotransposons found in the genomes of many rodents.
- The expression and function of IAP genes in normal and neoplastic cells are not fully understood.
Purpose of the Study:
- To investigate the transcription and expression of IAP genes in a murine plasmacytoma model.
- To identify and characterize IAP-specific RNA transcripts in MOPC-315 cells.
Main Methods:
- Hybridization analysis of RNA from MOPC-315 cells and normal mouse tissues with IAP gene probes.
- Northern blot analysis to identify and size IAP RNA transcripts.
- RNA synthesis inhibition assays using alpha-amanitin to determine the responsible RNA polymerase.
Main Results:
- IAP gene sequences hybridized to RNA from MOPC-315 cells, but not from normal tissues.
- Three major IAP RNA species (7.2, 5.3, and 3.8 kilobases) were detected in MOPC-315 cells.
- Higher levels of 7.2 and 5.3 kb transcripts were found in nuclear RNA compared to whole cell RNA.
- IAP RNA synthesis was initiated within the long terminal repeat and primarily utilized RNA polymerase II.
Conclusions:
- IAP gene expression is specific to the MOPC-315 plasmacytoma and absent in normal tissues.
- The regulation of IAP RNA processing, transport, or stability may differ between nuclear and whole cell fractions.
- RNA polymerase II is the key enzyme responsible for transcribing the primary IAP transcript.