Synthesis and distribution of primer RNA in nuclei of CCRF-CEM leukemia cells
- Bowman Gray School of Medicine of Wake Forest Univ., Winston-Salem, NC (USA)
The distribution of primer RNA and RNA-primed nascent DNA in nuclei of CCRF-CEM leukemia cells was examined, and the primer RNA purified from the nuclear matrices of these cells was characterized. RNA-primed nascent DNA was radiolabeled by incubating whole-cell lysates with ({alpha}-{sup 32}P)ATP and ({sup 3}H)dTTP in the presence of approximately physiological concentrations of the remaining ribo- and deoxyribonucleoside triphosphates. Nuclear subfactionation studies revealed that at least 94% of the primer RNA and RNA-primed nascent DNA were located within the insoluble matrix fraction of the nucleus. The predominant primer RNA isolated from the nuclear matrix was 8-10 nucleotides in length, and several lines of evidence indicated that this oligoribonucleotide was the functional primer RNA. Analysis of {sup 32}P transfer from ({alpha}-{sup 32}P)dTTP revealed a random distribution of ribonucleotides at the 3{prime}-end of the primer RNA. Data obtained from mixing experiments indicated that the association of RNA-primed nascent DNA with the nuclear matrix was not the result of aggregation of these fragments with the nuclear matrix. No significant amount of either primer RNA, RNA-primed nascent DNA, or phosphate transfer was detected in the high-salt-soluble (nonmatrix) fraction of the nucleus, although the nonmatrix fraction contained most of the newly replicated DNA. These observations provide evidence that in CCRF-CEM cells the synthesis of both primer RNA and RNA-primed Okazaki fragments takes place on the nuclear matrix, and that the primer RNA is degraded prior to the migration of the nascent DNA away from the matrix-bound DNA replication sites.
- OSTI ID:
- 6073878
- Journal Information:
- Biochemistry; (USA), Vol. 29:14; ISSN 0006-2960
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
DNA
AUTORADIOGRAPHY
RNA
SUBCELLULAR DISTRIBUTION
TUMOR CELLS
DNA REPLICATION
ATP
BIODEGRADATION
BIOSYNTHESIS
CELL NUCLEI
CENTRIFUGATION
CESIUM CHLORIDES
LEUKEMIA
PHOSPHORUS 32
TRITIUM COMPOUNDS
ALKALI METAL COMPOUNDS
ANIMAL CELLS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CELL CONSTITUENTS
CESIUM COMPOUNDS
CHEMICAL REACTIONS
CHLORIDES
CHLORINE COMPOUNDS
DAYS LIVING RADIOISOTOPES
DECOMPOSITION
DISEASES
DISTRIBUTION
HALIDES
HALOGEN COMPOUNDS
HEMIC DISEASES
HYDROGEN COMPOUNDS
IMMUNE SYSTEM DISEASES
ISOTOPES
LIGHT NUCLEI
NEOPLASMS
NUCLEI
NUCLEIC ACID REPLICATION
NUCLEIC ACIDS
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
PHOSPHORUS ISOTOPES
RADIOISOTOPES
SEPARATION PROCESSES
SYNTHESIS
550201* - Biochemistry- Tracer Techniques