Characterization of the 3' region of the human DNA topoisomerase I gene.
Previous studies suggest that topoisomerase I (Topo I) plays a critical role in cell growth. However, the structure of the Topo I gene has not yet been determined. Two complementary DNA (cDNA) clones for the human Topo I 4.1-kilobase mRNA were isolated independently from HeLa and KB cell cDNA libraries. These clones were identical and they contained 679 base pairs of coding and 1138 base pairs of noncoding sequences. The clones had a two-base difference in the 3' noncoding region compared to the Topo I cDNA from human placenta. The structure of the 3' end of the human Topo I gene from six human tumor cell lines was examined. The Topo I cDNA recognized 16.5, 24.2, and 16.0 kilobases of genomic DNA restricted with EcoRI, HindIII and PstI, respectively. The individual genomic fragments were ordered by double digestion and hybridization with cDNA subclones. digestion and hybridization with cDNA subclones. The results indicate that the human Topo I gene contains several intervening sequences. The gene arrangement was similar in all six cell lines and no polymorphism was observed. However, each digestion contained genomic fragments that hybridized with all the subclones, suggesting that at least one Topo I pseudogene, or another Topo I gene with a different structure, was present in every cell line. As predicted, double digestions generated at 161 base pair fragment that indicates the presence of an intronless pseudogene. In contrast to the DNA topoisomerase I gene, the presumptive pseudogene(s) appears to be hypomethylated. In addition to the 4.1-kilobase Topo I mRNA, a larger 6-kilobase RNA was identified in human KB and HeLa cells which could be a processed Topo I mRNA intermediate.
Zhou
B S
BS
Department of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill 27599-7365.
Bastow
K F
KF
Cheng
Y C
YC
D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D002460:Cell Line; D004264:DNA Topoisomerases, Type I / Q000235:genetics*; D005796:Genes; D006367:HeLa Cells / Q000201:enzymology; D006801:Humans; D008969:Molecular Sequence Data; D015183:Restriction Mapping; D014407:Tumor Cells, Cultured / Q000201:enzymology
D004264:DNA Topoisomerases, Type I