Isolation and expression of rat thymidylate synthase cDNA: phylogenetic comparison with human and mouse thymidylate synthases.
Two cDNA clones representing rat hepatoma thymidylate synthase (rTS) were isolated from a lambda ZAP II cDNA library using as a probe a fragment of the human TS cDNA. The two were identical except that one was missing 50 bp and the other 23 bp corresponding to the 5' coding region of the protein. The missing region was obtained by screening a rat genomic library. The open reading frame of rTS cDNA encoded 921 bp encompassing a protein of 307 amino acids with a calculated molecular mass of 35,015 Da. Rat hepatoma TS appears identical to normal rat thymus TS and the two sequences differ from mouse TS in the same eight amino acid residues. Six of these differences are in the first 21 amino acids from the amino-end. The human enzyme differed from rat and mouse TS at 17 residues where the latter two were identical, with most changes being conservative in nature. The three species differed completely at only four sites. Because the mouse TS shares four amino acids with human TS at sites which differ from rTS and a comparable situation does not exist between rTS and human TS, it is suggested that mouse TS is closer to human TS phylogenetically than rTS. The polymerase chain reaction was used to subclone the protein coding region of rTS into a high expression vector, which expressed rTS in Escherichia coli to the extent of 10 to 20% of its cellular protein. Although the amino-end of the amplified TS was unblocked, that isolated from a FUdR-resistant rat hepatoma cell line contained mostly N-acetylmethionine on its N-terminal end, a finding that may have significant regulatory consequences, which are discussed. The TS level in the resistant cell line was 60 to 70-fold higher than normal which was found to be associated with both multiple gene copies and an expanded TS mRNA pool.
Cieśla
J
J
Nencki Institute of Experimental Biology, Department of Cellular Biochemistry, Warsaw, Poland.
Weiner
K X
KX
Weiner
R S
RS
Reston
J T
JT
Maley
G F
GF
Maley
F
F
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular / Q000235:genetics; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000302:isolation & purification* / Q000378:metabolism; D004351:Drug Resistance; D004926:Escherichia coli / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics; D013940:Thymidylate Synthase / Q000235:genetics* / Q000378:metabolism; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D011994:Recombinant Proteins; D013940:Thymidylate Synthase