Molecular cloning of human A1S9 locus: an X-linked gene essential for progression through S phase of the cell cycle.
The temperature-sensitive (ts) A1S9 mouse L-cell mutant is defective in an X-linked gene essential for the progression of cells through the S phase of the cell duplication cycle. We recently reported the complementation of the ts A1S9 cell defect with total human DNA and the isolation of independent temperature-resistant transformants that retained a common set of human specific Alu-containing fragments. Here we describe the molecular cloning of these human DNA sequences from one of the secondary transformants. ST-1-0. A genomic library prepared from ST-1-0 was screened with a total human DNA probe, and two recombinant bacteriophages carrying overlapping segments were isolated. The cloned region was extended in both directions using a human X-chromosome specific library. In total, a human region spanning 42 kb in length, and containing all the Alu-specific DNA sequences found in ST-1-0, was isolated in five overlapping recombinant phages. The A1S9 gene appeared to be larger than the DNA recovered in individual phage isolates, as was assessed by transfection experiments. A single-copy probe derived from the phage DNA was shown to be conserved in independent primary, secondary, and tertiary transformants of ts A1S9 cells and mapped to the X chromosome by molecular hybridization. Northern blot hybridization of this probe with poly(A)+ mRNA derived from ST-1-0 cells identified a transcript of about 3.6 kb.
Zacksenhaus
E
E
Department of Microbiology, Faculty of Medicine, University of Toronto, Ontario, Canada.
Sheinin
R
R
D001435:Bacteriophages / Q000235:genetics; D015152:Blotting, Northern; D015139:Blotting, Southern; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D015342:DNA Probes; D004261:DNA Replication / Q000235:genetics; D015723:Gene Library; D008040:Genetic Linkage; D006801:Humans; D007399:Interphase / Q000235:genetics*; D015183:Restriction Mapping; D013696:Temperature; D014158:Transcription, Genetic; D014162:Transfection; D014960:X Chromosome
D015342:DNA Probes; D004247:DNA