Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.30.1 (S1 nuclease)
3,660 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The mouse thymidylate synthase (TS; EC 2.1.1.45) mRNA is unusual in that the poly(A) tail is added at the translation stop codon. To determine the sequence requirements for 3' processing of this mRNA, we constructed TS minigenes with deletion and point mutations in potential regulatory sequences. The minigenes were transiently transfected into cultured cells and the effect on 3' processing was determined by S1 nuclease protection assays. These analyses revealed that at least two elements are required for efficient polyadenylylation at the stop codon. The first is an upstream AUUAAA sequence. When this was changed to AUCAAA, polyadenylylation at the stop codon was blocked. However, when it was changed to the canonical AAUAAA hexanucleotide, the amount of TS mRNA increased severalfold. The second element is a stretch of 14 consecutive uridylate residues 32 nucleotides downstream of the stop codon. This U-rich region is absent from the human TS gene, which explains why the human TS mRNA is not polyadenylylated at the stop codon even though the two genes are otherwise almost identical through this region. The most surprising observation was that the U-rich region corresponds to the 3' end of a 360-nucleotide mouse L1 repetitive element that was inserted in opposite orientation to the gene more than 5 million years ago. Thus the polyadenylylation signal of the present mouse TS gene was created by the transposition of a repetitive element downstream of a cryptic polyadenylylation signal.
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PMID:Polyadenylylation signal of the mouse thymidylate synthase gene was created by insertion of an L1 repetitive element downstream of the open reading frame. 215 3

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.
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PMID: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. 253 45

Mouse thymidylate synthase minigenes that lack introns were constructed by ligating restriction fragments containing 4.5, 1.0, or 0.25 kilobase pairs (kb) of 5'-flanking DNA of the normal thymidylate synthase gene and as little as 0.25 kb of 3'-flanking DNA to full-length thymidylate synthase cDNA. All three minigenes were expressed at approximately the same levels following transfection into hamster V79 cells that were deficient in thymidylate synthase. S1 nuclease protection assays revealed that the multiple 5' and 3' termini of thymidylate synthase mRNA in cells transfected with these minigenes were at the same positions as those of the normal mRNA in mouse cells. Deletion analysis of the promoter region revealed that minigenes extending to position -150 nucleotides (relative to the AUG codon) were expressed at approximately the same level as those extending to -1 kb. However, minigenes extending to -53 nucleotides were inactive. To determine if the minigenes were capable of being regulated in a cell cycle-dependent manner, thymidylate synthase gene expression was measured in hamster cells that were stably transfected with the largest minigene and synchronized by serum-stimulation. Thymidylate synthase enzyme level and mRNA content increased 3-5-fold as cells progressed from G1 through S phase.
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PMID:Construction and expression of mouse thymidylate synthase minigenes. 282 52

As a part of a study of the role of bacteriophage T4-coded ribonucleoside diphosphate reductase in the switch-on and regulation of T4 DNA replication, we report the cloning and sequencing of the nrdA gene, coding for the alpha protein chain of the enzyme. The open reading frame of the nrdA gene begins 558 base pairs downstream of the 3' terminus of the td gene (thymidylate synthase). nrdB, encoding the beta chain of the enzyme, initiates 700 base pairs from the termination of nrdA. A high degree of similarity is found between the deduced amino acid sequence of the 754-residue alpha chain and the corresponding chain reported for nrdA of Escherichia coli; 56% of the residues are identical, with some segments reaching 84%. Some structural aspects of the derived alpha 2 subunit of the T4 enzyme are explored. By the S1 nuclease protection method, the RNA formed after T4 infection contains two prereplicative transcripts for nrdA, T3 and TU, and one postreplicative transcript, TL. T3 is found in low concentration. While the 5' termini of T3 and TL occur at sites near nrdA, TU apparently is a multicistronic transcript initiating farther upstream. The regulation of nrdA expression is examined in light of these findings.
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PMID:Total sequence, flanking regions, and transcripts of bacteriophage T4 nrdA gene, coding for alpha chain of ribonucleoside diphosphate reductase. 284 40

Previous studies have shown that thymidylate synthase gene expression is regulated over a wide range in response to growth stimulation in cultured mouse fibroblasts. In the present study we show that the gene is also regulated during the cell cycle in continuously growing cells. Our analyses were conducted with a fluorodeoxyuridine-resistant mouse 3T6 cell line that overproduces thymidylate synthase and its mRNA by a factor of 50 due to gene amplification. Cells were synchronized by mitotic selection. RNA blot analyses showed that the amount of thymidylate synthase mRNA increased 5- to 10-fold as cells progressed from G1 through the middle of S phase. S1 nuclease protection assays showed that the pattern of 5' termini of thymidylate synthase mRNA was the same in G1 and S phase. Despite the large increase in thymidylate synthase mRNA content, the level of the enzyme increased only by a factor of 2 as cells progressed from G1 to mid S phase. This apparent discrepancy can be explained by the fact that the enzyme is highly stable.
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PMID:Regulation of thymidylate synthase gene expression in mouse fibroblasts synchronized by mitotic selection. 291 7

Analysis of the sequence of cDNA corresponding to mouse thymidylate synthase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45) mRNA revealed that the termination codon TAA was followed immediately by a poly(A) sequence. This raised the possibility that mouse thymidylate synthase mRNA lacks a 3' untranslated region. In the present study, we have further investigated this possibility. DNA corresponding to the 3' end of the thymidylate synthase gene was isolated from a genomic library. The sequence of the genomic DNA was identical to that of the cDNA in the coding region. However, the termination codon was TAG in the genomic sequence rather than TAA, and poly(A) was not present in the genomic DNA. Sequences flanking the site of poly(A) addition were in good agreement with polyadenylylation consensus sequences. S1 nuclease analysis revealed that approximately 80% of the thymidylate synthase mRNA molecules were polyadenylylated at the termination codon. A secondary polyadenylylation site was detected 190-200 nucleotides downstream of the primary site. We conclude that the major species of mouse thymidylate synthase mRNA lacks a 3' untranslated region and that the final A of the termination codon is added by poly(A) polymerase. It appears that a 3' untranslated region is not essential for the accumulation or translation of this mRNA.
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PMID:Mouse thymidylate synthase messenger RNA lacks a 3' untranslated region. 302 94

The complete nucleotide sequence of a 1.8-kilobase DNA fragment containing the cell cycle-regulated thymidylate synthase gene (TMP 1) of the yeast Saccharomyces cerevisiae is presented. This analysis has revealed a 912-base pair open reading frame which encodes a 304-amino acid residue protein with a calculated Mr of 35,007. The tmp1-6 and cdc21-1 mutant alleles of this gene also have been sequenced, and both show single base pair changes which would result in different amino acid substitutions. The amino acid sequence of the yeast thymidylate synthase gene derived from the DNA sequence shows considerable homology when compared with the human, mouse, Herpesvirus saimiri, Leishmania major, Leishmania tropica, Escherichia coli, Lactobacillus casei, bacteriophage T4, and Bacillus subtilis phage phi 3T enzymes. Northern blot hybridization reveals that the TMP 1 mRNA is a 1.15-kilobase polyadenylated transcript. A set of consensus yeast mRNA splice sequences appears within the open reading frame of TMP 1, but S1 nuclease protection experiments reveal that splicing of the mRNA does not occur. Disruption of the gene by the introduction of a large insertion did not produce any defect besides the expected dependence on dTMP for growth. Specifically, the viability of the mutants in the presence of dTMP indicates that the protein does not play a significant structural role in a complex of replication enzymes.
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PMID:Molecular characterization of the cell cycle-regulated thymidylate synthase gene of Saccharomyces cerevisiae. 303 Oct 48

The 5' structure of mRNA transcribed from the dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene of the protozoan parasite Leishmania major has been characterized. S1 nuclease mapping identifies a heterogenous 5' structure which is not affected by growth phase or developmental stage. The DNA sequence of the 5' region of the DHFR-TS gene does not reveal homology with other trypanosomatid genes, eukaryotic consensus genetic elements, or the mammalian DHFR promoter element. This latter finding is especially significant as we show that the 5' region of the E. coli DHFR gene exhibits homology to the mammalian DHFR promoter element, despite their greater evolutionary distance.
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PMID:Sequence and S1 nuclease mapping of the 5' region of the dihydrofolate reductase-thymidylate synthase gene of Leishmania major. 355 43

We have isolated and analyzed the structure of the gene for thymidylate synthase from a 5-fluoro-2'-deoxyuridine-resistant 3T6 cell line that overproduces thymidylate synthase 50-fold by virtue of gene amplification. Three overlapping DNA segments containing the entire thymidylate synthase gene were identified in Charon 35 genomic libraries. Sequence analysis revealed that all of the coding regions were contained in a 12-kilobase segment of DNA. The gene has 6 introns ranging from 0.6 to 4.1 kilobases in length. The sequences at the 5' and 3' ends of each intron conformed to the consensus sequences for mammalian introns. S1 nuclease and primer extension assays showed that transcription of the thymidylate synthase gene initiates at several sites within a 66-nucleotide region. There are no TATAA or CCAAT sequences in the vicinity of the initiation sites. However, the region does contain DNA sequences that are known to stimulate binding of the transcription factors Sp1 and USF. These binding sites are adjacent to each other, suggesting that the two proteins may bind to the upstream region of the thymidylate synthase gene in a cooperative or competitive manner.
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PMID:Structure of the gene for mouse thymidylate synthase. Locations of introns and multiple transcriptional start sites. 378 3

The mechanism of expression of the structural gene (td) of T4 phage thymidylate synthase, which contains a 1,017-base pair intron, was studied by employing a coupled transcription-translation system with a td containing recombinant plasmid (pKTd2) as template. The [3H]leucine-labeled protein products synthesized in this system were treated with antibody to the synthase and the resulting immunoprecipitate was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two labeled polypeptides were obtained, one with an Mr of 32,000 and the other with an Mr of 25,000. The former corresponds in molecular weight to a subunit of T4-thymidylate synthase and the other to the 183-amino acid peptide encoded by exon I, the 5'-end of the interrupted td gene. When pKTd2 restricted in exon I was used as a template, labeled immunopeptides were not detected but, when restricted in the intron region or in exon II, only the 25,000 Mr exon I product was obtained. Both peptides (Mr = 25,000 and 32,000) were synthesized when the gene was restricted downstream to exon II. Active enzyme, as measured by the tritium release assay, was shown to form about 6 min after the td gene was added to the in vitro protein synthesizing system, and followed the appearance of mature mRNA, as evidenced by S1 nuclease protection studies. The enzyme increased linearly for another 14 min in conjunction with the appearance of the Mr = 32,000 immunopeptide. The exon I product, however, preceded the Mr = 32,000 peptide, indicating that a post-transcriptional processing event may be required for mature mRNA to be formed. Measurement of the RNA products from the td gene in a transcriptional system, with labeled probes from specific regions of the td gene, provided evidence in support of an RNA processing mechanism involving intron excision and exon splicing.
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PMID:In vitro expression of the intron-containing gene for T4 phage thymidylate synthase. 389 23


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