Cyclooxygenase-1 is a marker for a subpopulation of putative nociceptive neurons in rat dorsal root ganglia.

Immunocytochemical and morphometric techniques were used to quantify the distribution of cyclooxygenase (cox)-containing neurons in rat L5 dorsal root ganglia (DRG). Cox-1 immunolabelling was almost exclusively restricted to small diameter DRG neurons (< 1000 microm2), and was extensively colocalized with calcitonin gene-related peptide (CGRP) and isolectin B4 (IB4). Cox-1 was present in 65% and 70% of CGRP- and IB4-labelled neurons, respectively. Cox-1 labelling was also found in neurons expressing the sensory neuron-specific (SNS) Na+ channel. Cox-2 labelling was absent in DRG from normal rats. In the Freund's adjuvant model of monoarthritis, the proportion of cox-1-positive DRG neurons was unchanged and no neurons were found to be labelled for cox-2. In primary tissue culture, cox-1 immunolabelling persisted in vitro for up to 9 days and was present in morphologically identical neurons. The selective expression of cox-1 in peripheral ganglia was confirmed by the small number of nodose ganglion neurons and superior cervical ganglion (SCG) neurons labelled for cox-1. These data suggest that cox-1 is a marker for a subpopulation of putative nociceptive neurons in vitro and in vivo, and suggests that the prostaglandins synthesized by these neurons may be important for nociceptor function. These data may have important implications for the mode and mechanism of action of non-steroidal anti-inflammatory drugs (NSAIDs).

Base analogue mutagenesis in yeast and its modulation by pyrimidine deoxynucleotide pool imbalances: incorporation of bromodeoxyuridylate and iododeoxyuridylate.

Cells of the yeast, Saccharomyces cerevisiae, which are auxotrophic for thymidylate (tmpl) can also incorporate analogues of thymidylate. When the base analogue, 5-bromodeoxyuridylate, is incorporated into tmpl yeast cells it is lethal and mutagenic. Both lethality and mutation induction can be drastically altered by perturbation of the pyrimidine nucleotide pools. Analysis of mutation induction, bromodeoxyuridylate incorporation into DNA, and cell viability under various conditions revealed: (1) lethality and mutagenesis can be uncoupled, (2) thymidylate enhances mutagenesis and deoxycytidylate suppresses it, (3) mutation induction is not correlated with the magnitude of bromodeoxyuridylate incorporation into DNA. Therefore, in yeast, the pyrimidine nucleotide pools have a powerful effect on bromodeoxyuridylate mutagenesis. Both bromodeoxyuridylate and iododeoxyuridylate are extensively incorporated into the DNA of tmpl yeast cells; however, iododeoxyuridylate is non-mutagenic. Replication proceeds at the same rate in the presence of the natural substrate or either analogue. When cells are supplied with thymidylate and bromodeoxyuridylate together, there is no discrimination against bromodeoxyuridylate as a DNA precursor. However, in the presence of thymidylate and iododeoxyuridylate, there is a 3 to 1 discrimination against iododeoxyuridylate as compared to thymidylate.

Yeast/herpes simplex virus thymidine kinase gene fusions yield fusion proteins with thymidine kinase activity.

Expression in Saccharomyces cerevisiae of the Herpes Simplex Virus type-1 (HSV-1) thymidine kinase gene was accomplished by the construction of a gene fusion between the TK and a yeast gene. The fusion of yeast DNA sequences (which include a promoter and DNA that codes for the amino terminus end of the yeast gene product) with the TK gene resulted in a protein fusion with thymidine kinase activity.

Genetic and biochemical consequences of thymidylate stress.

We have examined the genetic and biochemical consequences of thymidylate stress in haploid and diploid strains of the simple eukaryote Saccharomyces cerevisiae (Bakers' yeast). Previously we reported that inhibition of dTMP biosynthesis causes "thymineless death" and is highly recombinagenic, but apparently not mutagenic, at the nuclear level; however, it is mutagenic for mitochondria. Concurrent provision of dTMP abolishes these effects. Conversely, excess dTMP is highly mutagenic for nuclear genes. It is likely that DNA strand breaks are responsible for the recombinagenic effects of thymidylate deprivation; such breaks could be produced by reiterative uracil incorporation and excision in DNA repair patches. In our experiments, thymidylate stress was produced both by starving dTMP auxotrophs for the required nucleotide and also by blocking de novo synthesis of thymidylate by various antimetabolites. We found that the antifolate methotrexate is a potent inducer of mitotic recombination (both gene conversion and mitotic crossing-over). This suggests that the gene amplification associated with methotrexate resistance in mammalian cells could arise, in part, by unequal sister-chromatid exchange induced by thymidylate stress. In addition, several sulfa drugs, which impede de novo folate biosynthesis, also have considerable recombinagenic activity.

Thymineless recombination in Saccharomyces cerevisiae is independent of the ability to undergo meiosis.

Thymine nucleotide starvation is recombinagenic in Saccharomyces cerevisiae and induces formation of the 'nuclear dense body', a structure characteristic of yeast cells in meiosis. Conceivably, thymineless recombination in yeast, presumed to be mitotic, might be meiotic in nature. We have tested this hypothesis and have found that thymineless recombination can be induced in strains incapable of meiotic exchange.

Nuclear morphology of yeast under thymidylate starvation.

During early meiotic development the yeast Saccharomyces cerevisiae has a characteristic nuclear dense body (NDB). It is shown that the NDB can also be induced in vegetatively growing cells through the inhibition of thymidylate synthetase which causes depletion of the dTMP pool and arrests DNA synthesis. The observations on NDBs and recombination levels suggest that thymidylate-stressed cells may activate parts of the meiotic pathway and, conversely, cells on sporulation medium may sense, among other things, reduced thymidylate levels and respond to the several stimuli by entering the meiotic pathway.

Induction of mitotic recombination in yeast by starvation for thymine nucleotides.

The biosynthesis of thymine nucleotides in Saccharomyces cerevisiae can be inhibited either by genetic lesions in the structural gene for thymidylate synthetase (TMP1) or by drugs that prevent the methylation of dUMP to dTMP. This methylation can be blocked by folate antagonists. We find that 5-fluoro-dUMP (FdUMP) is also an effective inhibitor in vivo. Inhibition of dTMP biosynthesis by these three different routes causes thymineless death. In addition to being cytotoxic, we find that FdUMP is highly recombinagenic in yeast but does not induce nuclear gene mutations. Provision of exogenous dTMP eliminates this induced mitotic recombination and cell killing. Similar results were obtained when a thymineless condition was provoked in cells by antifolate drugs or by dTMP deprivation in strains auxotrophic for this nucleotide. These findings show that, in contrast to the situation in prokaryotes, starvation for thymine nucleotides in yeast induces genetic recombination but is not mutagenic.

Isolation and characterization of yeast mutants auxotrophic for 2'-deoxythymidine 5'-monophosphate.

Mutant strains of Saccharomyces cerevisiae auxotrophic for deoxythymidine monophosphate (dTMP) were isolated and characterized. Two distinct classes of auxotrophs were obtained. One class had a simple requirement for dTMP and was analogous to thymine-requiring bacteria. The second class required dTMP, adenine, histidine and methionine and this complex nutritional phenotype was due to defects in folate metabolism. The dTMP-dependent growth of respiratory-competent grande auxotrophs was found to be markedly affected by media composition and carbon source. In the absence of dTMP thymineless death occurred in both mutant classes.

Genetic damage during thymidylate starvation in Saccharomyces cerevisiae.

Thymidylate starvation in a yeast mutant auxotrophic for dTMP caused cell death and the induction of mutations in the mitochondrial genome. After 24 h of starvation almost all surviving cells were respiratory deficient petites. In addition, shorter episodes of dTMP starvation induced chloramphenicol and erythromycin resistant mutants, indicating the occurrence of mitochondrial point mutations. Suboptimal concentrations of exogenous thymidylate were also found to induce petites and a decline in cell viability and the magnitude of these effects was acutely dependent upon the dTMP concentration. Cesium chloride gradient analysis of DNA from cells undergoing thymineless incubation revealed a progressive loss of mitochondrial DNA, and a decrease in the molecular weight of nuclear DNA.

Selection of yeast auxotrophs by thymidylate starvation.

A rapid procedure for the recovery of Saccharomyces cerevisiae auxotrophs was developed by exploiting the protection of these mutants from thymineless death when a required metabolite was withheld. The method can be used for thymidine 5'-monophosphate-requiring auxotrophs or wild-type strains blocked in de novo synthesis of thymidylate by folate antagonists.

Yeast mutants sensitive to trimethoprim.

Wild-type strains of Saccharomyces cerevisiae are resistant to growth inhibition by the folate antagonist trimethoprim. A mutant strain sensitive to trimethoprim was isolated. It was found to be sensitive to both ultraviolet light and X-irradiation. Genetic tests revealed that it was allelic with a known radiation-sensitive strain of Saccharomyces cerevisiae, rad 6-I. Strains harbouring a variety of mutant alleles conferring radiation-sensitivity were tested for sensitivity to trimethoprim. It was found that rad 6-I and each of the four known alleles of rad 18 conferred sensitivity to the drug, but all other rad mutants tested were trimethoprim-resistant. All trimethoprim-sensitive strains, including double mutants of rad 6 rad 18, gave rise to trimethoprim-resistant outgrowths at a rather high frequency (similar to 10-minus 5). Several resistant outgrowths were analysed. A wide variation in phenotype with respect of UV-sensitivty was found. Genetical analysis revealed that resistance to trimethoprim resulted from forward mutations at separate loci rather than back mutations of rad 6 or rad 18 alleles.

Thymineless death and ultraviolet sensitivity in Micrococcus radiodurans.

Thymine-requiring mutants of Micrococcus radiodurans have been isolated by selection on solid medium containing trimethoprim. Strains requiring either high concentrations of thymine (50 mug/ml) or low concentrations (2 mug/ml) for normal growth were obtained. The Thy(-) mutant requiring low thymine concentrations has been characterized. It was shown to retain the high ultraviolet light (UV) resistance typical of wild-type M. radiodurans, but it was not resistant to thymineless death. Preliminary exposure of the cells to thymineless conditions resulted in enhanced UV sensitivity, and this interaction occurred under conditions where "unbalanced growth" was inhibited by the addition of chloramphenicol. Upon addition of thymine to deprived cells, UV resistance was gradually restored, and this recovery took place in the absence of protein synthesis. A model is proposed to account for the similarity of thymineless death in bacteria whose deoxyribonucleic acid repair efficiencies differ widely.