Biomaterial Database

Autonomous replication of a DNA fragment containing the chromosomal replication origin of the human c-myc gene. 1990

C McWhinney, and M Leffak

Department of Biochemistry, Wright State University, Dayton, OH 45435.

The c-myc genes of HeLa cells are preferentially replicated in the transcriptional direction, from chromosomal origin sequences which display cell type-specific activity. Using a run-off replication assay involving in vitro extension of replication forks initiated in intact HeLa cells, bidirectional replication was observed to begin within a 3.5 kb domain 5' to the c-myc gene. To characterize the replication origin further a 2.4 HindIII-Xhol subfragment of the c-myc 5' flanking DNA was cloned in a selectable vector and transfected into HeLa cells. The resulting pNeo.Myc-2.4 construct persisted as a circular extrachromosomal element for more than 300 cell generations under selection, with recovery of approximately 500-1000 times the mass of plasmid initially introduced into the cells. Extrachromosomal circular pNeo.Myc-2.4 monomer was reisolated in supercoiled form, along with oligomeric and miniplasmid variants which had been generated in vivo; however, chromosomally integrated copies of the plasmid were not detectable in cultures containing extrachromosomal pNeo.Myc-2.4. The recovered pNeo.Myc-2.4 plasmid was resistant to Dpnl digestion and sensitive to Mbol digestion. After transfection with pNeo.Myc-2.4 BrUdR pulse labeling of long-term or short-term cultures demonstrated that the plasmid replicated semiconservatively, under controls similar to those imposed on chromosome replication. Bisection of the pNeo.Myc-2.4 insert suggested that c-myc 5' flanking DNA within 1.2 kb 5' to promoter P1 was sufficient to confer autonomously replicating sequence activity on the plasmid vector in transient replication assays.

UI	MeSH Term	Description	Entries
D008745	Methylation	Addition of methyl groups. In histo-chemistry methylation is used to esterify carboxyl groups and remove sulfate groups by treating tissue sections with hot methanol in the presence of hydrochloric acid. (From Stedman, 25th ed)	Methylations
D010957	Plasmids	Extrachromosomal, usually CIRCULAR DNA molecules that are self-replicating and transferable from one organism to another. They are found in a variety of bacterial, archaeal, fungal, algal, and plant species. They are used in GENETIC ENGINEERING as CLONING VECTORS.	Episomes,Episome,Plasmid
D011519	Proto-Oncogenes	Normal cellular genes homologous to viral oncogenes. The products of proto-oncogenes are important regulators of biological processes and appear to be involved in the events that serve to maintain the ordered procession through the cell cycle. Proto-oncogenes have names of the form c-onc.	Proto-Oncogene,Proto Oncogene,Proto Oncogenes
D002877	Chromosomes, Human	Very long DNA molecules and associated proteins, HISTONES, and non-histone chromosomal proteins (CHROMOSOMAL PROTEINS, NON-HISTONE). Normally 46 chromosomes, including two sex chromosomes are found in the nucleus of human cells. They carry the hereditary information of the individual.	Chromosome, Human,Human Chromosome,Human Chromosomes
D004247	DNA	A deoxyribonucleotide polymer that is the primary genetic material of all cells. Eukaryotic and prokaryotic organisms normally contain DNA in a double-stranded state, yet several important biological processes transiently involve single-stranded regions. DNA, which consists of a polysugar-phosphate backbone possessing projections of purines (adenine and guanine) and pyrimidines (thymine and cytosine), forms a double helix that is held together by hydrogen bonds between these purines and pyrimidines (adenine to thymine and guanine to cytosine).	DNA, Double-Stranded,Deoxyribonucleic Acid,ds-DNA,DNA, Double Stranded,Double-Stranded DNA,ds DNA
D004261	DNA Replication	The process by which a DNA molecule is duplicated.	Autonomous Replication,Replication, Autonomous,Autonomous Replications,DNA Replications,Replication, DNA,Replications, Autonomous,Replications, DNA
D004262	DNA Restriction Enzymes	Enzymes that are part of the restriction-modification systems. They catalyze the endonucleolytic cleavage of DNA sequences which lack the species-specific methylation pattern in the host cell's DNA. Cleavage yields random or specific double-stranded fragments with terminal 5'-phosphates. The function of restriction enzymes is to destroy any foreign DNA that invades the host cell. Most have been studied in bacterial systems, but a few have been found in eukaryotic organisms. They are also used as tools for the systematic dissection and mapping of chromosomes, in the determination of base sequences of DNAs, and have made it possible to splice and recombine genes from one organism into the genome of another. EC 3.21.1.	Restriction Endonucleases,DNA Restriction Enzyme,Restriction Endonuclease,Endonuclease, Restriction,Endonucleases, Restriction,Enzymes, DNA Restriction,Restriction Enzyme, DNA,Restriction Enzymes, DNA
D005822	Genetic Vectors	DNA molecules capable of autonomous replication within a host cell and into which other DNA sequences can be inserted and thus amplified. Many are derived from PLASMIDS; BACTERIOPHAGES; or VIRUSES. They are used for transporting foreign genes into recipient cells. Genetic vectors possess a functional replicator site and contain GENETIC MARKERS to facilitate their selective recognition.	Cloning Vectors,Shuttle Vectors,Vectors, Genetic,Cloning Vector,Genetic Vector,Shuttle Vector,Vector, Cloning,Vector, Genetic,Vector, Shuttle,Vectors, Cloning,Vectors, Shuttle
D006367	HeLa Cells	The first continuously cultured human malignant CELL LINE, derived from the cervical carcinoma of Henrietta Lacks. These cells are used for, among other things, VIRUS CULTIVATION and PRECLINICAL DRUG EVALUATION assays.	Cell, HeLa,Cells, HeLa,HeLa Cell
D006801	Humans	Members of the species Homo sapiens.	Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man