Identification of human gene complementing ts AlS9 mouse L-cell defect in DNA replication following DNA-mediated gene transfer

Abstract

The temperature-sensitive (ts) mouse L-cell, ts AlS9, is defective in a gene required for nuclear DNA replication early in the S phase of the cell cycle. Human DNA sequences were introduced into ts AlS9 cells together with the plasmid pSV2neo, which can confer resistance to the drug geneticin. Cotransformants, expressing both the plasmid-derived neomycin gene and the transferred human AlS9 gene, were selected for growth in the presence of the drug at the nonpermissive temperature (npt). The resulting transformants retained a common set of human-specific Alu repetitive DNA sequences. These are likely to be accommodated within, or in proximity to, the transferred human AlS9 gene. The results obtained provide the basis for cloning human genes required for DNA replication.

Molecular cloning, primary structure and expression of the human X linked A1S9 gene cDNA which complements the ts A1S9 mouse L cell defect in DNA replication.

“…At most, one colony out of 2.4 x 106 cells plated appeared in the control cultures. This is in agreement with the known reversion frequency of ts A1S9 cells at 39°C (Zacksenhaus and Sheinin, 1988). The transformation efficiency with pcAlS9-H2 was > 10-4.…”

“…A cDNA library was constructed in a eukaryotic expression vector from mRNA derived from ST-1-0, a temperatureresistant secondary transformant of ts AlS9 cells (Zacksenhaus and Sheinin, 1988). ST-1-0 was used as a source of RNA because it contains at least four copies of the human A1S9 gene in a mouse background.…”

“…This communication describes the molecular cloning and characterization of human cDNA clones which complement the ts A1S9 defect in DNA replication of mouse L cells. These cDNA clones were isolated using a genomic probe derived from the human AlS9 genomic locus which had previously been identified in, and subsequently isolated from, temperature-resistant transformants of ts AI S9 cells (Zacksenhaus and Sheinin, 1988. It is likely that these clones represent the human analogues of the ts AI S9 mouse gene and not a suppressor gene, because in both somatic cell fusion and transfection experiments a single complementing gene was identified and assigned independently to the short arm of the X chromosome Zacksenhaus et al, 1990).…”

“…The ts AlS9 mutant, the parental WT4 cells and the temperature-resistant, geneticin-resistant, secondary transformant of ts AlS9 cells, ST-1-0, were grown in aMEM medium containing 7.5 % fetal calf serum, as previously described (Zacksenhaus and Sheinin, 1988). The permissive temperature for the ts AlS9 cells is 34°C and the non-permissive temperature, 39°C.…”

“…We previously identified a human DNA region encompassing the A1S9 locus, which was retained in a number of independent, temperature-resistant transformants of ts A1S9 cells (Zacksenhaus and Sheinin, 1988). The entire locus was recovered from a genomic library prepared from a secondary transformant.…”