Localization of the synthesis of primer RNA on the nuclear matrix of CCRF-CEM leukemia cells
The biosynthesis of an RNA-primed DNA fragment is one of the key events involved in DNA replication. Current experimental evidence is consistent with the concept that DNA replication takes place at specific sites on the nuclear matrix, a residual framework of the interphase nucleus. The author's laboratory has recently found an enrichment of DNA primase activity and newly replicated DNA on the nuclear matrix of CCRF-CEM leukemia cells. The isolation of primer RNA has previously been difficult because of its low concentration within nuclei. However, since primer RNA was a specific marker of DNA replication forks, it seemed reasonable that primer RNA may be synthesized on the nuclear matrix. This prompted an examination into the distribution of primer RNA within nuclei of CCRF-CEM cells. RNA-primed nascent DNA was preferentially labeled by incubating whole cell lysates ({alpha}-{sup 32}P)ATP and ({sup 3}H) dTTP in the presence of approximately physiological concentrations of the remaining ribo- and deoxyribonucleoside triphosphates. Fractionation of the nuclei revealed that primer RNA and RNA-primed nascent DNA were almost exclusively localized within the high salt-insoluble matrix fraction of the nucleus. The primer RNA was purified by cesium chloride density gradient centrifugation and analyzed by HPLC gel filtration, polyacrylamide gel electrophoresis and phosphate transfer analysis. The predominant primer RNA isolated was 8-10 nucleotides in length and was covalently linked to nascent DNA as demonstrated by phosphate transfer analysis. No significant amount of RNA-primed nascent DNA or phosphate transfer was found in the nonmatrix fraction, even though it contained the majority of the nascent DNA. Serial dilution of both the matrix and nonmatrix samples indicated that the nuclear matrix contained at least 16-fold more primer RNA than the nonmatrix fraction.
- Research Organization:
- Bowman Gray School of Medicine, Winston-Salem, NC (USA)
- OSTI ID:
- 5613939
- Resource Relation:
- Other Information: Thesis (Ph. D.)
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
DNA REPLICATION
BIOLOGICAL PATHWAYS
RNA
BIOSYNTHESIS
RNA POLYMERASES
ENZYME ACTIVITY
BIOLOGICAL LOCALIZATION
BIOLOGICAL MARKERS
CELL NUCLEI
ELECTROPHORESIS
LEUKEMIA
LIQUID COLUMN CHROMATOGRAPHY
NUCLEOTIDES
TRACER TECHNIQUES
TRITIUM COMPOUNDS
TUMOR CELLS
ULTRACENTRIFUGATION
ANIMAL CELLS
CELL CONSTITUENTS
CENTRIFUGATION
CHROMATOGRAPHY
DISEASES
ENZYMES
HYDROGEN COMPOUNDS
IMMUNE SYSTEM DISEASES
ISOTOPE APPLICATIONS
NEOPLASMS
NUCLEIC ACID REPLICATION
NUCLEIC ACIDS
NUCLEOTIDYLTRANSFERASES
ORGANIC COMPOUNDS
PHOSPHORUS-GROUP TRANSFERASES
POLYMERASES
SEPARATION PROCESSES
SYNTHESIS
TRANSFERASES
550201* - Biochemistry- Tracer Techniques
550901 - Pathology- Tracer Techniques