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Biomedical subjects

L F Johnson

Publications and source records attributed to L F Johnson.

At least 19 recordsLinked to original sources
PubMed2000

Inhibition of the mammalian transcription factor LSF induces S-phase-dependent apoptosis by downregulating thymidylate synthase expression.

The thymidylate synthase (TS) gene, which is induced at the G(1)-S transition in growth-stimulated cells, encodes an enzyme that is essential for DNA replication and cell survival. Here we demonstrate that LSF (LBP-1c, CP2) binds to sites within the TS promoter and intronic regions that are required for this induction. Mutation of the LSF binding sites inhibits G(1)-S induction of mRNA derived from a TS minigene. Furthermore, expression of dominant-negative LSF (LSFdn) prevents the increase in TS enzyme levels during G(1)-S, and induces apoptosis in growth- stimulated mouse and human cell lines. Such apoptosis can be prevented either by circumventing the TS requirement through addition of low concentrations of thymidine, or by coexpression of the TS gene driven by a heterologous promoter. Induction of apoptosis by LSFdn parallels the process known as thymineless death, which is induced by the TS inhibitor and chemotherapeutic drug 5-fluorodeoxyuridine. Thus, LSF is a novel regulatory factor that supports progression through S-phase by targeting a single gene that is critical for cell survival.

Amino Acid Sequence↗
PubMed1999

Complex transcriptional initiation pattern of the thymidylate synthase promoter in mouse tissues.

Thymidylate synthase (TS) is an essential enzyme that must be expressed in all proliferating cells. The mouse TS promoter lacks a TATA box and an initiator element and initiates transcription over a broad region in cultured fibroblasts. The goal of this study was to determine if expression of the TS gene in a variety of cells and tissues involves the use of alternative promoters or different patterns of transcriptional initiation sites. The amount of TS mRNA and the pattern of initiation sites were determined using S1 nuclease protection assays. We found that even though the amount of TS mRNA varied over a wide range, reflecting differences in cell proliferation rates, the pattern of initiation sites was nearly identical in all of the cell lines and tissues that were examined. Therefore transcription of the TS gene is directed by a single promoter that is capable of being expressed in a wide variety of cellular environments.

Aging↗
PubMed1999

Inactivation of MED-1 elements in the TATA-less, initiator-less mouse thymidylate synthase promoter has no effect on promoter strength or the complex pattern of transcriptional start sites.

The mouse thymidylate synthase (TS) promoter is a member of a family of promoters that lack a TATA box as well as an initiator element and that initiate transcription at many sites over a broad initiation window. An element (MED-1) downstream of the initiation window of almost all promoters of this family has been proposed to be important for promoter activity, as well as for multiple start site utilization. Two consensus MED-1 elements are located downstream of the initiation window of the TS promoter. To determine the role of the MED-1 elements in the TS promoter, one or both elements were inactivated by site-directed mutagenesis and the effects on promoter function were determined. We found that inactivation of the MED-1 elements had no measurable effect on promoter strength, the boundaries of the initiation window, or the pattern of transcriptional start sites. Furthermore, inactivation of the elements did not affect the ability of the TS promoter to direct S phase-specific expression of the gene in growth-stimulated cells. We conclude that the MED-1 element does not play a significant role in TS promoter function and therefore is not an essential component of all TATA-less promoters with complex transcriptional initiation patterns.

Animals↗
PubMed1998

Analysis of transcripts derived from sequences upstream of the bidirectional mouse thymidylate synthase promoter.

The promoter of the mouse thymidylate synthase (TS) gene lacks a TATAA box and an initiator element and is capable of directing transcriptional initiation with approximately equal strength and over broad initiation windows in both directions. The goal of the present study was to determine if the TS promoter directs the transcription of a second gene that is upstream of the TS gene by characterizing the transcripts that correspond to the upstream sequences. RNA blot analyses revealed the presence of 1.4 and 5 kb cytoplasmic, polyadenylated transcripts that include sequences upstream of the TS promoter. The transcripts were much more abundant in a cell line in which the TS gene is amplified. S1 nuclease protection assays showed that the transcripts have multiple 5' termini. An exon trap approach identified a potential splice donor site that might correspond to the 3' end of the first exon of the upstream gene. A cDNA library was probed with a sequence from the putative first exon, and six different cDNA clones were isolated. However, analysis of the sequences of the cDNAs revealed that the upstream transcripts were not spliced at the potential 3' donor site but instead extended into a repetitive LINE (long interspersed nuclear element) sequence that begins 0.3 kb upstream of the TS promoter. RNase protection assays confirmed that the in vivo transcripts extend into the LINE element. Therefore it appears that the upstream transcripts are unlikely to correspond to a functional mRNA molecule.

Amino Acid Sequence↗
PubMed1998

Pre-mRNA processing enhancer (PPE) element increases the expression of an intronless thymidylate synthase gene but does not affect intron-dependent S phase regulation.

The pre-mRNA processing enhancer (PPE) element is an RNA sequence element derived from the intronless HSV-TK gene. Insertion of the element into the highly intron-dependent human beta-globin gene leads to efficient expression in the absence of splicing. We have analyzed the effect of the PPE element on the expression of mouse thymidylate synthase (TS) minigenes. We have previously shown that the expression of intronless TS minigenes is moderately (up to 20-fold) stimulated by the inclusion of introns. Furthermore, S phase-specific expression of TS minigenes in growth-stimulated cells depends on the presence of a spliceable intron as well as the TS promoter. The goal of our study was to determine if the PPE element would overcome the dependence on introns for efficient expression and for S phase-specific expression of transfected TS minigenes. We found that insertion of the PPE element into an intronless TS minigene partially overcame intron dependence. However, the increase in expression was much less than that observed for the intronless beta-globin gene. We also found that intronless TS or HSV-TK genes that contained the PPE element and that were driven by the TS promoter were expressed at a constant level in serum-stimulated cells. However, when an intron was included in these genes, they were expressed in an S phase-specific manner. Thus the PPE element was not able to overcome the dependence on introns for S phase-specific expression of TS minigenes.

Animals↗
PubMed1998

Responses to different levels of esophageal acidification during waking and sleep.

The purpose of this study was to assess the effect of hydrogen ion concentration of intraesophageal infusions during sleep on acid clearance time and latency to swallow and arousal responses from sleep. We studied 10 normal volunteers during sleep via polysomnography and concomitant esophageal pH monitoring. Sleep prolonged the acid clearance time of both pH 3.0 and 1.2. Swallow and arousal latencies were both progressively decreased with decreasing pH of the infusate (P < 0.05, 0.07, respectively). We concluded that intraluminal hydrogen ion concentration creates an afferent warning stimulus that produces prompt airway-protective responses.

Adult↗
PubMed1998

Medical miracles.

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Drug Interactions↗
PubMed1997

Inhibition of mouse thymidylate synthase promoter activity by the wild-type p53 tumor suppressor protein.

The p53 tumor suppressor protein is an important negative regulator of the G1 to S transition in mammalian cells. We have investigated the effect of p53 on the expression of the mouse thymidylate synthase (TS) gene, which normally increases as cells enter S phase. A luciferase indicator gene that was driven by the wild-type or various modified forms of the TATA-less mouse TS promoter was transiently cotransfected with a p53 expression plasmid into TS-deficient hamster V79 cells and the level of luciferase activity was determined. We found that wild-type p53 inhibited TS promoter activity by greater than 95% but had a strong stimulatory effect on an artificial promoter that contained multiple p53-binding sites. In contrast, an expression plasmid that encodes a mutant form of p53 or a wild-type retinoblastoma tumor suppressor protein had little effect on TS promoter activity. Deletion of sequences upstream or downstream of the TS essential promoter region, or inactivation of each of the known elements within the essential promoter region, had no effect on the ability of wild-type p53 to inhibit TS promoter activity. Our observations indicate that the inhibition of TS promoter activity by p53 is not due to the presence of a specific p53 negative response element in the TS promoter. Rather, it appears that p53 inhibits the TS promoter by sequestering ("squelching") one or more general transcription factors.

Animals↗
PubMed1996

Splicing signals are required for S-phase regulation of the mouse thymidylate synthase gene.

The thymidylate synthase (TS) gene is expressed at a much higher level in cells undergoing DNA replication than in nondividing cells. In growth-stimulated mammalian cells, TS mRNA content increases 10 to 20-fold as cells progress from G1 through S phase. However, the rate of transcription of the TS gene does not increase during this interval, indicating that the gene is regulated at the posttranscriptional level. We have shown that both the promoter of the mouse TS gene and TS introns are necessary (although neither is sufficient) for S-phase-specific regulation of TS mRNA content. In the present study, we examined in more detail the role of introns in regulating TS mRNA levels in growth-stimulated cells. TS minigenes that contain normal or modified introns were stably transfected into mouse 3T6 fibroblasts, and the regulation of the minigenes was compared with that of the endogenous TS gene. TS minigenes that contain TS intron 1 or 2 maintain S-phase regulation. Deletion of most of the interior of the introns had only minor effects on regulation. However, when splicing of the intron was inhibited by alteration of the splice donor and acceptor sites, the minigene was expressed at a constant level following growth stimulation. Minigenes consisting of the TS promoter linked to either a luciferase or a human beta-globin indicator gene were growth regulated when spliceable introns were included in the minigenes. However, when the introns were eliminated, the minigenes were expressed at a constant level. These observations indicate that the splicing reaction itself, rather than a control sequence within the intron, is important for growth-regulated expression of the TS gene. Possible mechanisms to account for the dual requirement for the TS promoter and intron splicing for proper regulation of the TS gene are discussed.

Animals↗