Often employees provide a false economy--saving time, balancing schedules, and then moving on to the next task. This motive is well intentioned, however misguided.

Equipment Safety↗

PubMed1995

Regulation of mouse thymidylate synthase gene expression in growth-stimulated cells: upstream S phase control elements are indistinguishable from the essential promoter elements.

Expression of the mammalian thymidylate synthase (TS) gene in growth-stimulated cells is closely coordinated with entry into S phase. Previous studies with transfected TS minigenes have shown that sequences upstream of the coding region as well as an intron in the transcribed region are both necessary for proper regulation of TS mRNA content in growth-stimulated cells. The goal of the present study was to identify the upstream regulatory elements. Minigenes consisting of TS 5' flanking sequences linked to the TS coding region (interrupted by introns 1 and 2) were stably transfected into mouse 3T6 cells. Deletion and site-directed mutagenesis of the 5' flanking region revealed that there is a close correspondence between the upstream sequences that are necessary for S phase regulation and the 30 nucleotide region that is essential for promoter activity. These observations raised the possibility that regulation of the TS gene occurs at the transcriptional level. However, nuclear run-on assays showed that the rate of transcription of the TS gene changed very little during the G1-S phase transition. Furthermore, when the TS promoter was linked to an intron-less luciferase indicator gene, there was no change in expression following growth-stimulation. Therefore it appears that the TS gene is controlled primarily at the posttranscriptional level, and that the TS essential promoter region is necessary (although not sufficient) for proper S phase regulation.

Animals↗

PubMed1995

Effect of cigarette smoking on gastroesophageal reflux measured by 24-h ambulatory esophageal pH monitoring.

OBJECTIVE: We reassessed the effect of cigarette smoking on gastroesophageal reflux because two previous ambulatory 24-h pH monitoring studies showed equivocal results and did not relate heartburn to changes in pH reflux events. METHODS: Our protocol design considered nicotine's pharmacokinetic half-life; 14 smokers with heartburn and esophagitis abstained from smoking for 48 h before and during an ambulatory 24-h esophageal pH monitoring study (24-h pH). After resuming their smoking habits for 48 h or more, they underwent a second 24-h pH study and smoked 20 regular, filtered Marlboro cigarettes. Acid reflux was defined as a drop in intraesophageal pH to a value < 4 at 5 cm above the lower esophageal sphincter and was measured as percent exposure and reflux events (total N, those > or = 5 min, and longest event). Heartburn episodes were noted by the patients and were correlated later to acid reflux events. RESULTS: Cigarette smoking significantly increased the percentage time that the pH was < 4 during a 24-h period from 7.35 to 11.1% (medians; p < 0.007). This increased exposure occurred predominantly during the day while in the upright posture and resulted from significant increases in both reflux events and those parameters that measure acid clearance (T events > or = 5 min and longest event). While smoking, the patients noted a 114% increase in daytime heartburn episodes that immediately followed a pH reflux event (3.5 to 7.5 episodes, medians; p < 0.009). CONCLUSIONS: Smoking 20 cigarettes has a greater effect on acid reflux and heartburn than purported.

Adult↗

PubMed1994

Bidirectional promoter of the mouse thymidylate synthase gene.

The promoter of the mouse thymidylate synthase (TS) gene lacks both a TATAA box and an initiator element and directs transcriptional initiation at multiple sites over a 90 nucleotide initiation window. Earlier studies defined an essential region near the 5' end of the initiation window that is required for promoter activity. The essential region contains possible binding sites for Sp1 and Ets transcription factors. In the present study we show that this essential region stimulates transcription with approximately equal strength in both directions. Transcription is initiated over a broad initiation window in the reverse direction. The same elements are important for the reverse promoter and for the normal TS promoter. Sequences upstream of the essential region partially suppress expression in the reverse direction. The TS 5' flanking region, in either the normal or inverted orientation, directs S phase-specific expression of a TS minigene. This raises the possibility that an upstream gene and the TS gene may be coordinately induced at the G1/S phase boundary by a common set of control elements.

Amino Acid Sequence↗

PubMed1994

In vitro transcription of the TATAA-less mouse thymidylate synthase promoter: multiple transcription start points and evidence for bidirectionality.

The mouse thymidylate synthase (TS) promoter (pTS) lacks a TATAA box and an initiator element, and has multiple transcription start points (tsp) located across a 90-bp region. We have developed an in vitro transcription system for pTS using circular templates and nuclear extracts from HeLa cells or mouse 3T6 fibroblasts. The amount of RNA synthesized and the locations of the tsp were determined by S1 nuclease protection assays. The transcription system reproduced the complex pattern of in vivo tsp, except that the downstream tsp were used preferentially. The reaction temperature, concentrations of DNA template and MgCl2, and incubation time were optimized. The pTS core region contains binding sites for the Sp1 and Ets transcription factors. Inactivation of the Sp1-binding element led to a twofold reduction in transcription and a preferential use of upstream tsp. Inactivation of the Ets-binding element, which reduced promoter activity tenfold in vivo, had only a minor effect in vitro. Addition of a strong initiator element introduced a new tsp, but did not eliminate the complex tsp pattern. To determine if pTS had bidirectional promoter activity, the promoter was inverted and analyzed for transcriptional activity. The inverted promoter was found to initiate transcription at multiple tsp and had approximately the same strength as the normal pTS.

Animals↗

PubMed1994

Posttranscriptional regulation of thymidylate synthase gene expression.

Thymidylate synthase (TS) is an essential enzyme that catalyzes the formation of thymidylic acid in the de novo biosynthetic pathway and is the target enzyme for a variety of chemotherapeutic agents. The TS gene is expressed at a much higher level in proliferating cells than in quiescent cells. Control is primarily exerted at the posttranscriptional level. Studies with chimeric TS minigenes have shown that regulation of TS mRNA content in growth-stimulated mouse fibroblasts requires the presence of sequences located upstream of the essential promoter elements. In addition, an efficiently spliced intron must be present within the transcript. Neither sequence by itself is sufficient for proper regulation, suggesting that the upstream and downstream sequences may communicate to effect regulation. A possible mechanism by which the upstream sequences influence the efficiency of splicing of TS transcripts in a cell cycle specific manner is described. Expression of the human TS gene is also controlled at the translational level. The TS enzyme is able to block the translation of its own mRNA by binding to the message in the vicinity of the AUG start codon. The translational block is relieved in the presence of substrates or inhibitors of the enzyme. The autogenous translational regulation of TS mRNA is likely to be responsible for the rapid increase in TS enzyme level that occurs when cells are exposed to certain TS inhibitors. Elucidation of the mechanism by which the translational control is exerted may lead to the design of more effective TS inhibitors.

Animals↗

PubMed1994

Upright refluxers without esophagitis differentiated from bipositional refluxers with esophagitis by simultaneous manometry and pH monitoring conducted in two postures before and after a meal.

OBJECTIVE: To determine whether two dissimilar groups characterized by 24-h esophageal pH monitoring would have individual reflux events that occur under the same or different circumstances when challenged by a meal and monitored in different postures. These groups consisted of upright refluxers without esophagitis (n = 10) versus biopositional refluxers with esophagitis (n = 8). METHODS: Our evaluation consisted of a questionnaire completed prior to simultaneous manometry and pH monitoring. This monitoring was conducted over a 120-min period that incorporated the upright and recumbent postures both before and after a meal. Three of our four criteria for reflux were independent of a classic pH event. RESULTS: From the questionnaire, it was apparent that dyspeptic symptoms almost always occurred in the upright refluxers. In addition, provocation for their heartburn had atypical characteristics regarding posture, and their degree of regurgitation was intensified by the postprandial state. During dual monitoring, the upright posture and the meal provoked an increase in frequency of reflux in the upright refluxers, as opposed to only recumbency in the bipositional refluxers. Moreover, reflux events in the upright refluxers usually were associated with Valsalva maneuvers and were recognized as symptoms, two features that were less true in the bipositional refluxers. A similar percentage of reflux events occurred over a low basal lower esophageal sphincter pressure and after a lower esophageal sphincter relaxation, in both groups. CONCLUSION: The two groups manifested distinctively different reflux characteristics, presumably due to dissimilar mechanisms.

Adult↗

PubMed1993

Stimulation of gene expression by introns: conversion of an inhibitory intron to a stimulatory intron by alteration of the splice donor sequence.

Efficient expression of many mammalian genes depends on the presence of at least one intron. We previously showed that addition of almost any of the introns from the mouse thymidylate synthase (TS) gene to an intronless TS minigene led to a large increase in expression. However, addition of intron 4 led to a reduction in minigene expression. The goal of the present study was to determine why TS intron 4 was unable to stimulate expression. Insertion of intron 4 into an intron-dependent derivative of the ribosomal protein L32 gene did not lead to a significant increase in expression, suggesting that its inability to stimulate expression was due to sequences within the intron. Deleting most of the interior of intron 4, improving the putative branch point, removing purines from the pyrimidine stretch at the 3' end of the intron, or removing possible alternative splice acceptor or donor sites within the intron each had little effect on the level of expression. However, when the splice donor sequence of intron 4 was modified so that it was perfectly complementary to U1 snRNA, the modified intron 4 stimulated expression approximately 6-fold. When the splice donor site of TS intron 1 (a stimulatory intron) was changed to that of TS intron 4, the modified intron 1 was spliced very inefficiently and lost the ability to stimulate mRNA production. Our observations support the idea that introns can stimulate gene expression by a process that depends directly on the splicing reaction.

Animals↗

PubMed1993

Introns are essential for growth-regulated expression of the mouse thymidylate synthase gene.

The thymidylate synthase (TS) gene is expressed at much higher levels in proliferating cells than in quiescent cells. We have been studying the sequences that are important for regulating the mouse TS gene. We previously showed that DNA sequences upstream of the essential promoter elements as well as downstream of the ATG codon are both necessary (but neither is sufficient) for normal regulation in growth-stimulated cells. In the present study, we examined the possible roles of the coding region, polyadenylation signal, and introns as downstream regulatory elements. Minigenes consisting of 1 kb of the TS 5'-flanking region, the coding region (with or without various introns at their normal locations), and polyadenylation signals from the TS gene, the human beta-globin gene, and the bovine growth hormone gene were stably transfected into wild-type mouse 3T6 cells. Minigenes that contained introns 5 and 6, 1 and 2, or 1 alone were regulated regardless of which polyadenylation signal was included. A minigene that contained an internally deleted version of intron 1 was also regulated in response to growth stimulation. However, when all introns were omitted, there was little if any change in the level of minigene expression as cells progressed from G1 through S phase. These observations indicate that TS introns contain sequences that are necessary for normal growth-regulated expression of the mouse TS gene. These sequences appear to be associated with sequences that are important for splicing and to function in cooperation with upstream regulatory elements to bring about normal S-phase-specific expression.

Animals↗

PubMed1993

Lack of an initiator element is responsible for multiple transcriptional initiation sites of the TATA-less mouse thymidylate synthase promoter.

The mouse thymidylate synthase promoter lacks a TATA box and initiates transcription at many sites across a 90-nucleotide initiation window. We showed previously that wild-type promoter activity is maintained with a promoter that extends only 13 nucleotides upstream of the first start site. G/A-rich and G/C-rich promoter elements were identified in the vicinity of the first transcriptional start site. The goals of the present study were to determine whether there are additional promoter elements in the initiation window and to determine why transcription initiates across such a broad region. Minigenes containing a variety of substitution, deletion, and insertion mutations in the promoter region were transfected into cultured cells, and the effects on expression and the pattern of start sites were determined. The results indicate that there are no additional promoter elements downstream of the G/C box. The boundaries of the transcription window are established by elements near the 5' end of the window, whereas the pattern of start sites is determined by sequences within the window. The promoter lacks an initiator element. When an initiator element was inserted, transcription initiated predominantly at the position directed by the initiator when it was inserted within the initiation window but not when it was inserted immediately upstream of the window.

Animals↗

PubMed1992

G1 events and the regulation of genes for S-phase enzymes.

The genes for S-phase enzymes are expressed at low levels in quiescent mammalian cells but at high levels during DNA replication. Regulation occurs at multiple levels by mechanisms that are different for each gene. Current research is focused on identifying the control elements and trans-acting factors for each gene and establishing relationships between these regulatory mechanisms and the G1 signal transduction pathway.

Animals↗

PubMed1991

Multiple protein-DNA interactions in the TATAA-less mouse thymidylate synthase promoter.

The mouse thymidylate synthase (TS) promoter lacks a TATAA box and directs transcriptional initiation at multiple sites over a 60 nucleotide region. All of the sequences that are important for transcription are located within close proximity to the first initiation site. Gel mobility shift and footprinting analyses with various sequences from this region of the TS promoter identified three major protein-DNA interactions. One of these corresponds to Sp1 interacting with a nonconsensus binding site downstream of the first transcriptional initiation site. Inactivation of the binding site by site-directed mutagenesis led to a 3-fold reduction in gene expression as well as a significant change in distribution of transcriptional start sites. The proteins responsible for the other two complexes (CII and CIII) do not appear to correspond to any of the common transcription factors that have been studied previously. The CII protein binds very close to the first initiation site. Inactivation of the binding site by site-directed mutagenesis had little effect on expression. The CIII protein binds immediately upstream of CII. Inactivation of this site led to a 12-fold reduction in expression, indicating that it is important for expression of the TS gene.

Animals↗

PubMed1991

Purification and characterization of recombinant mouse thymidylate synthase.

Recombinant mouse thymidylate synthase (TS) expressed at high levels in Escherichia coli was purified to homogeneity in greater than 70% yield by a rapid three-step procedure. Both 0.1% Triton X-100 and 10% glycerol were required to stabilize the enzyme whose activity remained unchanged after 1 month when stored at -20 degrees C. Thermal inactivation of the enzyme was a first-order process at 37 degrees C, with t1/2 values of 6.9, 15.6 and 3.0 min at pH 5.5, 7.0 and 8.5, respectively. The presence of saturating levels of dUMP at pH 8.5 increased the t1/2 of inactivation of 38 min. The pH profile for enzyme activity showed a narrow optimum region centered at pH 7.0, which was mirrored by the shape of the Km, dUMP/Vmax plot. The pH dependence of Kd for the covalent inhibitory ternary complex of enzyme, 5-fluoro-2'-deoxyuridylate and 5,10-methylenetetrahydrofolate exhibited a broad minimum between pH 5.5 and 8.5, and ranged between 3.1, 0.8 and 1.1 nM at pH 5.5, 7.0 and 8.5, respectively. The UV/VIS spectrum of the native enzyme exhibited a maximum at 280 nm (epsilon = 98,200 M-1 cm-1), while that of the inhibitory ternary complex showed an additional maximum at 320 nm. The 19F-NMR spectrum of the mouse enzyme:FdUMP binary complex revealed two new resonances at -2.8 and -34.8 ppm. The most deshielded resonance represented the noncovalent binary complex while the other resonance was assigned to the nucleotide covalently bound to the enzyme. The alteration of nucleotide binding equilibria produced by addition of H4 folate was exemplified by both an increase in intensity and a 5 ppm deshielding of the resonance attributed to the covalent FdUMP-enzyme complex. Addition of formaldehyde to the latter mixture produced the covalent ternary complex which resulted in the collapse of the resonances at -2.8 and -39.5 ppm and the appearance of a new resonance at -12.4 ppm.

Animals↗

PubMed1991

Simultaneous esophageal pH monitoring and scintigraphy during the postprandial period in patients with severe reflux esophagitis.

To compare reflux events detected by intraesophageal pH monitoring with that of scintigraphy, we simultaneously performed both techniques along with esophageal manometry in nine patients with severe reflux esophagitis. Two hundred eighteen reflux events were detected in the recumbent posture after a meal during a 40-min interval. Both techniques simultaneously detected only 23% of all reflux events. Scintigraphy alone detected 61% of all reflux events as opposed to 16% for pH monitoring. Of those reflux events diagnosed only by scintigraphy, more occurred while the intraesophageal pH was less than 4 (ie, during an acid-clearing interval) than while the intraesophageal pH was greater than 4 (ie, when intragastric contents were neutralized by the meal). Most reflux events occurred during periods of stable, but low LES pressure. While reflux events diagnosed by scintigraphy significantly decreased during the second of two 20-min postprandial intervals, those by pH monitoring tended to increase. That simultaneous scintigraphy and pH monitoring agreed on less than 1/3 of all reflux events not only underscores the fact that both techniques measured different physical components of the esophageal refluxate (ie, volume vs acid concentration, respectively), but also were influenced by different physiologic events such as the ingestion of a meal, gastric emptying, and esophageal acid clearance.

Adult↗