Autonomous replication of human chromosomal DNA fragments in human cells.
AUTOR(ES)
Masukata, H
RESUMO
We have examined whether a human chromosome has distinct segments that can replicate autonomously as extrachromosomal elements. Human 293S cells were transfected with a set of human chromosomal DNA fragments of 8-15 kilobase pairs that were cloned on an Escherichia coli plasmid vector. The transfected cells were subsequently cultured in the presence of 5-bromodeoxyuridine during two cell generations, and several plasmid clones labeled in both of the daughter DNA strands were isolated. Efficiency of replication of these clones, as determined from the ratios of heavy-heavy and one-half of heavy-light molecules to total molecules recovered from density-labeled cells, was 9.4% per cell generation on the average. Replication efficiency of control clones excluded during the selection was about 2.2% and that of the vector plasmid alone was 0.3%. A representative clone p1W1 replicated in a semiconservative manner only one round during the S phase of the cell cycle. It replicated extrachromosomally without integration into chromosome. The human segment of the clone was composed of several subsegments that promoted autonomous replication at different efficiencies. Our results suggest that certain specific nucleotide sequences are involved in autonomous replication of human segments.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=275748Documentos Relacionados
- Analysis of the autonomous replication behavior in human cells of the dihydrofolate reductase putative chromosomal origin of replication.
- Evaluation of autonomous plasmid replication in transfected mammalian cells.
- Autonomous replication of a DNA fragment containing the chromosomal replication origin of the human c-myc gene.
- Autonomous replication in human cells of multimers of specific human and bacterial DNA sequences.
- Initiation of replication in chromosomal DNA induced by extracts from proliferating cells.