Human thymidylate synthase gene: isolation of phage clones which cover a functionally active gene and structural analysis of the region upstream from the translation initiation codon.
Two genomic DNA fragments partially encoding human thymidylate synthase (TS) [EC 2.1.1.45] were previously cloned in lambda phage from the mouse cell transformant, but had no transforming activity on mouse TS-negative mutant cells. In this study, an additional genomic DNA for human TS was cloned and demonstrated to have the transforming activity in combination with one of the two previously cloned DNAs and to produce human TS mRNA. The two transforming genomic DNAs overlapped and covered a region of 23 kb in total. Using fragments from one of these DNAs, the structure of the 1.2-kb region around the ATG initiator codon of the TS gene was analyzed in relation to regulatory sequences of the gene. Sequence determination demonstrated the presence of an unusual inverted repeat consisting of a triple tandem repeat of a 28-bp sequence and an inverted sequence of the same length. These sequences can form three possible, stable, stem-loop structures, which may be interconvertible. Based on S1 nuclease mapping data and a line of circumstantial evidence, we deduced two major mRNA cap sites within the inverted sequence. Comparison of the human and mouse sequences upstream from the ATG initiator codon revealed many significant blocks of sequence homology, especially in the regions around the deduced cap sites.
Takeishi
K
K
Department of Immunology and Virology, Saitama Cancer Center Research Institute.
Kaneda
S
S
Ayusawa
D
D
Shimizu
K
K
Gotoh
O
O
Seno
T
T
D000818:Animals; D010582:Bacteriophage lambda / Q000201:enzymology*; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D003062:Codon; D004279:DNA, Viral / Q000032:analysis / Q000235:genetics; D005814:Genes, Viral; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D010442:Peptide Chain Initiation, Translational; D014176:Protein Biosynthesis; D012333:RNA, Messenger; D012367:RNA, Viral / Q000032:analysis / Q000235:genetics; D013940:Thymidylate Synthase / Q000235:genetics*; D014170:Transformation, Genetic
D003062:Codon; D004279:DNA, Viral; D012333:RNA, Messenger; D012367:RNA, Viral; D013940:Thymidylate Synthase