Biomedical subjects

S B Haase

Publications and source records attributed to S B Haase.

3 recordsLinked to original sources

PubMed1991

Replication control of autonomously replicating human sequences.

Three autonomously replicating plasmids carrying human genomic DNA and a vector derived from Epstein-Barr virus were studied by density labelling to determine the number of times per cell cycle these plasmids replicate in human cells. Each of the plasmids replicated semi-conservatively once per cell cycle. The results suggest that these human autonomously replicating sequences undergo replication following the same controls as chromosomal DNA and represent a good model system for studying chromosomal replication. We also determined the time within the S phase of the cell cycle that three of the plasmids replicate. Centromeric alpha sequences, which normally replicate late in S phase when in their chromosomal context, were found to replicate earlier when they mediate replication on an extrachromosomal vector. Reproducible patterns of replication within S phase were found for the plasmids, suggesting that the mechanism specifying time of replication may be subject to experimental analysis with this system.

Bromodeoxyuridine↗

PubMed1989

Isolation of human sequences that replicate autonomously in human cells.

We have isolated a heterogeneous collection of human genomic sequences which replicate autonomously when introduced into human cells. The novel strategy for the isolation of these sequences involved cloning random human DNA fragments into a defective Epstein-Barr virus vector. This vector alone was unable to replicate in human cells, but appeared to provide for the nuclear retention of linked DNA. The human sequences persist in a long-term replication assay (greater than 2 months) in the presence of the viral nuclear retention sequences. Using a short-term (4-day) assay, we showed that the human sequences are able to replicate in the absence of all viral sequences. The plasmids bearing human sequences were shown to replicate based on the persistence of MboI-sensitive plasmid DNA in the long-term assay and the appearance of DpnI-resistant DNA in the short-term assay. The human sequences were shown to be responsible for the replication activity and may represent authentic human origins of replication.

Base Sequence↗

PubMedDate not supplied

Improved EBV shuttle vectors.

Shuttle vectors based on Epstein-Barr virus (EBV) replicate autonomously in the nuclei of human cells. These vectors represent reasonable models for chromosomes, have low background mutation frequencies, and have been useful for studying induced mutation in human cells. Two improvements in the EBV vector system are discussed. Attempts are described to increase vector copy number per cell by using a limited period of replication driven by the simian virus 40 (SV40) origin of replication. Isolation of human sequences that can replace the viral origin of replication in providing for autonomous replication of the vectors is also described. These improvements are leading toward shuttle vectors that are more efficient and more closely resemble authentic chromosomes.

Cells, Cultured↗