Somatic Cell and Molecular Genetics, Vot. 18, No. 5, 1992, pp. 409-415
Regulatory Sequences Clustered at the 5' End of the First Intron of the Human Thymidylate Synthase Gene Function in Cooperation with the Promoter Region Sumiko Kaneda] Nobuyuki Horie, 2 Keiiehi Takeishi, 2 Atsushi Takayanagi, 1 Takeshi Seno, 1 and Dai Ayusawa 3 1Laboratory of Mutagenesis, National Institute of Genetics, 1111 Yata, Mishima 411; 2School of Food and Nutritional Sciences, lJ)~iversityof Shizuoka, 52-1 Yada, Shizuoka 422; and 3Institute of Applied Microbiology, Universityof Tokyo, Bunkyo-ku, 7bkyo 113, Japan Received 15 June 1992--Final t7 August 1992
Abstract--A human thymidylate synthase (TS) minigene containing 5'- and Y-flanking sequences, all the exons, and only intron 1 showed a normal frequency of stable transformation when transfected into TS-negative mutant cells, whereas minigenes in which intron 1 was replaced by intron 2 or deleted in the above construct showed only a few percent of the above frequency. Introduction of intron 1 into the above intronless or intron 2 minigene restored the transforming activities regardless of its position and orientation. Deletion analysis revealed two positive and one negative regulatory sequences in the 5' end of intron 1, each of which seemed to bind specific proteins as shown by gel shift analysis. Intron 1 also stimulated expression of a TS promoter-CAT gene construct but not that of an SV40 promoter-CA T gene construct. These results' indicate that the multiple regulatory sequences clustered in intron 1 stimulate TS gene expression in concert with the 5'-flanking sequences.
INTRODUCTION
Thymidylate synthase (TS) (EC2.1.1.45) plays an essential role in regulating the balanced supply of the four DNA precursors for normal DNA replication and is involved in various biological and genetic abnormalities (1-6). The expression of the TS gene is cell cycle-regulated not only at the level of the enzymatic activity (7), but also at the level of the mature mRNA mainly due to posttranscriptional events (8, 9). We have isolated ?t phage clones containing the functionally active hmnan TS gene (10) and determined the complete nucleotide sequence (11). The biologically active unit spans about 16 kilobases (kb) and is
composed of seven exons and six introns (11). The major transcriptional start sites are located between - 179 and - 160 nucleotides upstream from the ATG initiation codon. A cluster of an Alu sequence appears in the region upstream of nucleotide -529. Between the transcription start sites and the Atu cluster, there is no typical promoter sequence such as the TATA, CAin.T, or GC box (11). However, the promoter was located within about 400 base pairs (bp) upstream from the ATG codon by transfection assay with the use of the X phage genomic clones and a TS minigene construct, each with deletions of various lengths in their 5'flanking sequences (10, 11). The promoter located by transient expression assay of the
409
0740-7750/92/0700-0409506.50/0 © 1992 Plenum Publishing Corporation
410
CAT gene ligated to the various 5'-flanking sequences was consistent with that described above (10, 11). In the above transfection experiments (11), we used a particular minigene designated as pmHTS-1 that contained both the 5'- and 3'-flanking sequences, all the exons, and only intron 1 at its original position. This minigene appeared to function quite normally with respect to cell cycle-dependent regulation and promoter function (11). Thus, we took advantage of using pmHTS-1 to search for a regulatory sequence in the human TS gene. We describe here the presence of multiple regulatory sequences, clustered in the 5' half of intron 1 and their several unique features.
MATERIALS AND METHODS
Construction of TS Minigenes. The minigene pmHTS-1 consists of 3.7 kb of the 5'-flanking sequence upstream from the ATG codon, 1.7 kb of the 3'-flanking sequence downstream from the polyadenylation site, all the exons, and only intron 1 at its original position (Fig. 1) (11). For construction of an intronless minigene (pmHTS-0), a 1.9-kb SphI-ApaI fragment containing intron 1 was cut out from pmHTS-1 and a 200-bp SphI-ApaI fragment from a cDNA clone pcHTS-1 (12) was inserted into the same position. For construction of a minigene containing only intron 2 (pmHTS-2), a 200-bp EcoRI-ApaI fragment was cut out from pmHTS-0, and a 2.6-kb of EcoRI-ApaI fragment containing intron 2 from a X genomic clone (10) was inserted into the same position. Deletion mutant clones of pmHTS-1 were made by cutting out appropriate fragments of intron 1 from pmHTS-1 followed by religation. Insertion mutants of pmHTS-0 and pmHTS-2 were constructed by introducing a 780-bp SphI-BamHI fragment, which contains the 5' half sequence of intron 1 into
Kaneda et al.
A pmHTS-1
~
Transforming activity (%) lOO 13
Epp
pmHTS-O pmHTS-2
•'-O
III
2
"1
B pmHTS-1 pmHTS-1-AAB3 pmHTS-1-ABB11 pmHTS-1-AAN6 pmHTS-I-ANB8 pmHTS-1-AANh9
c
pmHTS-0
lOO
Regulatory Sequences Clustered at the 5' End of the First Intron of the Human Thymidylate Synthase Gene Function in Cooperation with the Promoter Region Sumiko Kaneda] Nobuyuki Horie, 2 Keiiehi Takeishi, 2 Atsushi Takayanagi, 1 Takeshi Seno, 1 and Dai Ayusawa 3 1Laboratory of Mutagenesis, National Institute of Genetics, 1111 Yata, Mishima 411; 2School of Food and Nutritional Sciences, lJ)~iversityof Shizuoka, 52-1 Yada, Shizuoka 422; and 3Institute of Applied Microbiology, Universityof Tokyo, Bunkyo-ku, 7bkyo 113, Japan Received 15 June 1992--Final t7 August 1992
Abstract--A human thymidylate synthase (TS) minigene containing 5'- and Y-flanking sequences, all the exons, and only intron 1 showed a normal frequency of stable transformation when transfected into TS-negative mutant cells, whereas minigenes in which intron 1 was replaced by intron 2 or deleted in the above construct showed only a few percent of the above frequency. Introduction of intron 1 into the above intronless or intron 2 minigene restored the transforming activities regardless of its position and orientation. Deletion analysis revealed two positive and one negative regulatory sequences in the 5' end of intron 1, each of which seemed to bind specific proteins as shown by gel shift analysis. Intron 1 also stimulated expression of a TS promoter-CAT gene construct but not that of an SV40 promoter-CA T gene construct. These results' indicate that the multiple regulatory sequences clustered in intron 1 stimulate TS gene expression in concert with the 5'-flanking sequences.
INTRODUCTION
Thymidylate synthase (TS) (EC2.1.1.45) plays an essential role in regulating the balanced supply of the four DNA precursors for normal DNA replication and is involved in various biological and genetic abnormalities (1-6). The expression of the TS gene is cell cycle-regulated not only at the level of the enzymatic activity (7), but also at the level of the mature mRNA mainly due to posttranscriptional events (8, 9). We have isolated ?t phage clones containing the functionally active hmnan TS gene (10) and determined the complete nucleotide sequence (11). The biologically active unit spans about 16 kilobases (kb) and is
composed of seven exons and six introns (11). The major transcriptional start sites are located between - 179 and - 160 nucleotides upstream from the ATG initiation codon. A cluster of an Alu sequence appears in the region upstream of nucleotide -529. Between the transcription start sites and the Atu cluster, there is no typical promoter sequence such as the TATA, CAin.T, or GC box (11). However, the promoter was located within about 400 base pairs (bp) upstream from the ATG codon by transfection assay with the use of the X phage genomic clones and a TS minigene construct, each with deletions of various lengths in their 5'flanking sequences (10, 11). The promoter located by transient expression assay of the
409
0740-7750/92/0700-0409506.50/0 © 1992 Plenum Publishing Corporation
410
CAT gene ligated to the various 5'-flanking sequences was consistent with that described above (10, 11). In the above transfection experiments (11), we used a particular minigene designated as pmHTS-1 that contained both the 5'- and 3'-flanking sequences, all the exons, and only intron 1 at its original position. This minigene appeared to function quite normally with respect to cell cycle-dependent regulation and promoter function (11). Thus, we took advantage of using pmHTS-1 to search for a regulatory sequence in the human TS gene. We describe here the presence of multiple regulatory sequences, clustered in the 5' half of intron 1 and their several unique features.
MATERIALS AND METHODS
Construction of TS Minigenes. The minigene pmHTS-1 consists of 3.7 kb of the 5'-flanking sequence upstream from the ATG codon, 1.7 kb of the 3'-flanking sequence downstream from the polyadenylation site, all the exons, and only intron 1 at its original position (Fig. 1) (11). For construction of an intronless minigene (pmHTS-0), a 1.9-kb SphI-ApaI fragment containing intron 1 was cut out from pmHTS-1 and a 200-bp SphI-ApaI fragment from a cDNA clone pcHTS-1 (12) was inserted into the same position. For construction of a minigene containing only intron 2 (pmHTS-2), a 200-bp EcoRI-ApaI fragment was cut out from pmHTS-0, and a 2.6-kb of EcoRI-ApaI fragment containing intron 2 from a X genomic clone (10) was inserted into the same position. Deletion mutant clones of pmHTS-1 were made by cutting out appropriate fragments of intron 1 from pmHTS-1 followed by religation. Insertion mutants of pmHTS-0 and pmHTS-2 were constructed by introducing a 780-bp SphI-BamHI fragment, which contains the 5' half sequence of intron 1 into
Kaneda et al.
A pmHTS-1
~
Transforming activity (%) lOO 13
Epp
pmHTS-O pmHTS-2
•'-O
III
2
"1
B pmHTS-1 pmHTS-1-AAB3 pmHTS-1-ABB11 pmHTS-1-AAN6 pmHTS-I-ANB8 pmHTS-1-AANh9
c
pmHTS-0
lOO
