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Analysis of a replication initiation sequence from the adenosine deaminase region of the mouse genome
V J Virta-Pearlman1, P H Gunaratne, A C Chinault
1Institute for Molecular Genetics, Baylor College of Medicine, Houston, Texas 77030.
Abstract:
A 4-kb HindIII fragment that supported the efficient autonomous replication of plasmid vector pDY-, a replication-defective construct based on Epstein-Barr virus sequences, in human K562 cells was rescued from amplified double-minute chromosomes containing the murine adenosine deaminase locus. Polymerase chain reaction assays of size-fractionated nascent strands demonstrated that replication initiation occurred within the same 1- to 2-kb region of this fragment in autonomously replicating plasmids containing the sequence in either orientation, in double-minute chromosomes, and in the single-copy locus at its normal chromosomal location. The complete sequence of this fragment was determined; it contains a 248-bp polypurine tract and consensus binding site sequences for several putative transcription and replication factors.
Insights
Researchers identified a specific DNA fragment that enables autonomous replication in human cells. This fragment, containing a polypurine tract, initiates replication consistently across different genomic contexts, including plasmids and chromosomes.
Area of Science:
- Molecular Biology
- Genetics
- Epigenetics
Background:
- Autonomous replication is crucial for maintaining extrachromosomal DNA elements like plasmids.
- Understanding replication origins is key to gene expression and genome stability.
Purpose of the Study:
- To identify and characterize a DNA fragment that drives autonomous replication in human cells.
- To determine the precise region and orientation-dependent initiation of replication within this fragment.
Main Methods:
- Rescue of a replication-supporting fragment from amplified double-minute chromosomes.
- Polymerase chain reaction (PCR) assays on size-fractionated nascent DNA strands.
- DNA sequencing of the identified fragment.
Main Results:
- A 4-kb HindIII fragment from murine adenosine deaminase locus supported efficient autonomous replication in K562 cells.
- Replication initiation was localized to a 1- to 2-kb region within the fragment.
- Initiation occurred regardless of sequence orientation in plasmids, double-minute chromosomes, and single-copy loci.
Conclusions:
- The identified fragment contains an origin of replication active in human cells.
- A 248-bp polypurine tract and consensus factor binding sites within the fragment are likely involved in replication initiation.