Enhanced thymidine uptake causes the lowered thymidine requirement of D. discoideum auxotroph HPS 401

Dictyostelium discoideum strain HPS 401 contains a spontaneous mutation that lowers the amount of thymidine required for cell growth relative to that of the auxotrophic parental strain HPS 400. Growth studies in defined medium show that as little as 8 micrograms thymidine/ml supports maximal growth of HPS 401, whereas 50 micrograms/ml is required by HPS 400. In contrast, both strains require over 40 micrograms thymidylate/ml to achieve maximal growth. HPS 401 exhibits thymidineless death when grown without thymidine; relative viability decreases to less than 0.01% after 190 h incubation. Assays for enzymes related to thymidine metabolism reveal that none of the strains tested (HPS 401, HPS 400, and prototrophic HPS 83 cells) contain detectable thymidine phosphorylase activity and that the specific activity of thymidine kinase is the same in these three strains. Thin-layer chromatography of extracts from cells grown on radiolabeled thymidine shows that there is no detectable conversion of thymidine to thymine in any of these strains. These analyses show that HPS 401 has rapid intracellular accumulation of thymidine, while only slight uptake is observed with HPS 400 or wild-type strains. HPS 401 also shows greater uptake of uridine in comparison to HPS 400 and wild-type cells. Thymidylate uptake was the same for all three strains. Thus, the mutation giving rise to the HPS 401 phenotype selectively increases the uptake of thymidine into the cell, where it can be efficiently utilized for DNA synthesis by the "salvage" pathways of nucleotide metabolism.     

Permanent or reversible conjugation of 2'-O- or 5'-O-aminooxymethylated nucleosides with functional groups as a convenient and efficient approach to the modification of RNA and DNA sequences

2'-O-Aminooxymethyl ribonucleosides are prepared from their 3',5'-disilylated 2'-O-phthalimidooxymethyl derivatives by treatment with NH(4)F in MeOH. The reaction of these novel ribonucleosides with 1-pyrenecarboxaldehyde results in the efficient formation of stable and yet reversible ribonucleoside 2'-conjugates in yields of 69-82%. Indeed, exposure of these conjugates to 0.5 M tetra-n-butylammonium fluoride (TBAF) in THF results in the cleavage of their iminoether functions to give the native ribonucleosides along with the innocuous nitrile side product. Conversely, the reaction of 5-cholesten-3-one or dansyl chloride with 2'-O-aminooxymethyl uridine provides permanent uridine 2'-conjugates, which are left essentially intact upon treatment with TBAF. Alternatively, 5'-O-aminooxymethyl thymidine is prepared by hydrazinolysis of its 3'-O-levulinyl-5'-O-phthalimidooxymethyl precursor. Pyrenylation of 5'-O-aminooxymethyl thymidine and the sensitivity of the 5'-conjugate to TBAF further exemplify the usefulness of this nucleoside for modifying DNA sequences either permanently or reversibly. Although the versatility and uniqueness of 2'-O-aminooxymethyl ribonucleosides in the preparation of modified RNA sequences is demonstrated by the single or double incorporation of a reversible pyrenylated uridine 2'-conjugate into an RNA sequence, the conjugation of 2'-O-aminooxymethyl ribonucleosides with aldehydes, including those generated from their acetals, provides reversible 2'-O-protected ribonucleosides for potential applications in the solid-phase synthesis of native RNA sequences. The synthesis of a chimeric polyuridylic acid is presented as an exemplary model.     

Recombinogenic activity of nalidixic acid for artificial hybrids but not for natural strains of Candida albicans: evidence for the monoploidy of natural strains

Nalidixic acid induces segregation of auxotrophs from prototrophic hybrids of Candida albicans artifically produced by fusing complementing auxotrophic protoplasts. The auxotrophies recovered are limited to those introduced through the fusion process, and patterns of segregations for linked auxotrophic markers demonstrate the segregants are products mitotic crossing-over. Nalidixic acid does not induce auxotrophies of any sort in clinical isolates of C. albicans. These findings are contrary to a current hypothesis that natural strains of C. albicans are diploid and heterozygous for a variety of auxotrophic mutations.     

Availability of bases and nucleosides as precursors of nucleic acids in L cells and in the agent of meningopneumonitis

Tribby, Ilse I. E. (University of Chicago, Chicago, Ill.), and James W. Moulder. Availability of bases and nucleosides as precursors of nucleic acids in L cells and in the agent of meningopneumonitis. J. Bacteriol. 91:2362-2367. 1966.-Uninfected L cells and the meningopneumonitis agent propagated in L cells utilized exogenous adenine, guanine, and their ribonucleosides and deoxyribonucleosides for synthesis of both deoxyribonucleic acid (DNA) and ribonucleic acid. Cytosine, cytidine, and uridine were also incorporated into the nucleic acids of both host and parasite. L cells and the meningopneumonitis agent incorporated uracil, thymine, and deoxyuridine very poorly. L cells utilized thymidine and deoxycytidine almost exclusively for DNA synthesis, but the meningopneumonitis agent did not incorporate these nucleosides at all. Since the L cell had previously been shown to convert added thymidine to its nucleotides, mainly the triphosphate, it was concluded that the meningopneumonitis agent can utilize neither the thymidine nor the thymidine nucleotides of the L-cell pool, and that it can probably synthesize the thymidine triphosphate needed for DNA synthesis from the uridine of the L-cell pool.     

Pectinolytic activity of revertants of auxotrophic strains of Aspergillus niger

From conidia of 4 different auxotrophic A. niger strains 400 spontaneous revertants (100 from each strain) were obtained, and in one case additionally 100 revertants induced by mutagens (UV+NTG). The revertants showed a considerable differentiation with regard to the total pectinolytic activity. Its highest increase occurred in revertants originating from auxotrophs greatly predisposed to synthesize pectinases. In the case of revertants induced by mutagenes an increase in the frequency of their formation was observed, as well as an increased participation of revertants with higher pectinolytic activity compared to both their initial auxotrophic and prototrophic strain.     

Inhibition of DNA synthesis by griseolutein in Escherichia coli through a possible interaction at the cell surface.

Griseolutein acts as a bactericidal agent, its toxicity decreasing with increase in cell density. There is no evidence that griseolutein acts at a specific stage of cell cycle. Inhibition of incorporation of radioactive thymidine into acid-insoluble fraction of cells is marked and rapid, while inhibition of incorporation of uridine also takes place. Incorporation of 32Pi into the acid-insoluble fraction of cells is inhibited while the incorporation into the nucleotide pool is not. Concentration of any of the four deoxyribonucleoside triphosphates in griseolutein-treated cells are similar to or higher than those in untreated cells. No extensive degradation of cellualr DNA is caused by griseolutein. DNA synthesis in plasmolyzed cells in not inhibited by griseolutein.     

Nanotube-mediated cross-feeding couples the metabolism of interacting bacterial cells

Bacteria frequently engage in cross-feeding interactions that involve an exchange of metabolites with other micro- or macroorganisms. The often obligate nature of these associations, however, hampers manipulative experiments, thus limiting our mechanistic understanding of the ecophysiological consequences that result for the organisms involved. Here we address this issue by taking advantage of a well-characterized experimental model system, in which auxotrophic genotypes of E. coli derive essential amino acids from prototrophic donor cells using intercellular nanotubes. Surprisingly, donor–recipient cocultures revealed that the mere presence of auxotrophic genotypes was sufficient to increase amino acid production levels of several prototrophic donor genotypes. Our work is consistent with a scenario, in which interconnected auxotrophs withdraw amino acids from the cytoplasm of donor cells, which delays feedback inhibition of the corresponding amino acid biosynthetic pathway and, in this way, increases amino acid production levels. Our findings indicate that in newly established mutualistic associations, an intercellular regulation of exchanged metabolites can simply emerge from the architecture of the underlying biosynthetic pathways, rather than requiring the evolution of new regulatory mechanisms.     

Reversible inhibition by human serum lipoproteins of cell proliferation

Normal human serum or plasma was studied for the presence of inhibitors of cell proliferation by assaying inhibition of incorporation of labeled thymidine into acid-insoluble fraction using human FL cells. Lipoprotein fraction obtained by gel filtration through Sepharose 4B and by KBr density gradient centrifugation was found to play a major part of the inhibitory activity of the serum. It was also shown that the inhibitory activity resides in low-density lipoprotein (LDL). The addition of the lipoprotein fraction to growing FL cells caused an early decrease in the transport of uridine and thymidine across the membrane. This change in the permeability of membrane was followed by the preferential inhibition of DNA synthesis and a reduction in the percentage of mitotic cells in the cell population. The inhibition of the growth was reversible and was observed in various types of cells irrespective of species.     

Alteration in de novo pyrimidine biosynthesis during uridine reversal of pyrazofurin-inhibited DNA synthesis

Pyrazofurin, a pyrimidine nucleoside analogue with antineoplastic activity, inhibits cell proliferation and DNA synthesis in cells by inhibiting uridine 5'-phosphate (UMP) synthase. It has been previously shown in concanavalin A (con A)-stimulated guinea pig lymphocytes (23) that pyrazofurin-inhibited DNA synthesis could be selectively reversed by exogenous uridine (Urd). In this report, we have examined possible mechanisms for the Urd reversal with experiments that determine the ability of exogenous Urd to (a) interfere with either the intracellular transport of pyrazofurin, or the conversion of pyrazofurin to its intracellularly active form, pyrazofurin-5'-phosphate; (b) reverse the pyrazofurin block of [14C]orotic acid incorporation into DNA; and (c) alter the pattern of exogenous [3H]Urd incorporation into DNA-thymine (DNA-Thy) and DNA-cytosine (DNA-Cyt) during pyrazofurin inhibition of pyrimidine de novo biosynthesis. The results of these experiments showed that Urd reversal does not occur through altered pyrazofurin transport or intracellular conversion to pyrazofurin-5'-phosphate, nor does it alter the distribution of [3H]Urd in DNA-Thy and DNA-Cyt. Instead, these findings indicate that the primary mechanism for exogenous Urd reversal of pyrazofurin inhibition of DNA synthesis involves the reversal of pyrazofurin inhibition of UMP synthase, thus restoring orotic acid incorporation into lymphocyte DNA through the pyrimidine de novo pathway.     

Construction of a broad host range cosmid cloning vector and its use in the genetic analysis of Rhizobium mutants

We have constructed a cosmid derivative of the low copy-number broad host-range cloning vector pRK290 (Ditta et al., 1980) by inserting a 1.6-kb Bg/II fragment containing lambda cos into the unique Bg/II site in pRK290. The new vector, pLAFR1, is 21.6 kb long, confers tetracycline resistance, contains a unique EcoRI site, and can be mobilized into and stably replicates within many Gram-negative hosts. We constructed a clone bank of Rhizobium meliloti DNA in pLAFR1 using a partial EcoRI digest. The mean insert size was 23.1 kb. When the clone bank was mated (en masse) from Escherichia coli to various R. meliloti auxotrophic mutants, tetracycline-resistant (Tcr) transconjugants were obtained at frequencies ranging from 0.1 to 0.8, and among these, prototrophic colonies were obtained at frequencies ranging from 0.001 to 0.007. pLAFR1 cosmids were mobilized from R. meliloti prototrophic colonies into E. coli and then reintroduced into R. meliloti auxotrophs. In most cases, 100% of these latter Tcr transconjugants were prototrophic.     

Auxotrophy of Pseudomonas aeruginosa in cystic fibrosis

Seventy-four of 403 (18.4%) sputum isolates of Pseudomonas aeruginosa from 49 of 136 (36.0%) adults with cystic fibrosis (CF) were auxotrophic mutants. Two of 11 (18.2%) isolates of P. aeruginosa taken from patients with non-CF bronchiectasis were also auxotrophic. All 99 strains taken from non-bronchiectatic sources were prototrophic. Forty-six of 55 (83.6%) CF auxotrophs required one or more of 36 growth factors tested; the requirements for the remaining 9 isolates were not identified. Methionine was the sole factor required by 17 of 22 (77.3%) isolated which depended on a single factor. We conclude that auxotrophy is a feature of P. aeruginosa infection in cystic fibrosis.     

A cytological analysis of the antimetabolite activity of 5-hydroxyuracil in Vicia faba roots

The effects of 5-hydroxyuracil (5-HU) (isobarbituric acid) upon cell elongation, mitosis, and DNA synthesis were studied in Vicia faba roots. 5-HU had no consistent effect upon root elongation. It blocked DNA synthesis (analyzed by photometric measurements of Feulgen dye in nuclei) during the first 6 hours of treatment; the block spontaneously disappeared by the 12th hour of treatment. Uracil and thymine had no effect upon this block of synthesis. Both thymidine and uridine reversed the block in 6 and 9 hours respectively. In all cases blockage of DNA synthesis was followed by inhibition of mitosis (determined by changes in the percentage of cells in mitosis) and resumption of DNA synthesis was followed by resumption of mitosis. Inhibition indices calculated from the mitotic data indicated a competitive relationship between 5-HU and thymidine and 5-HU and uridine. 5-HU is considered to block DNA synthesis by competing with thymidine for sites on enzymes involved in the synthesis. It is suggested that uridine reverses the block in synthesis by undergoing a conversion to thymidine.     

The nature of arginine auxotrophy in cutaneous populations of staphylococci.

L-Arginine was required for growth by a high percentage of strains of Staphylococcus species that were niche-specific and/or host-specific, but was usually not required for growth by species showing a wide host range. Growth stimulation patterns with arginine intermediates indicated that most of the auxotrophic strains had blocks in an early step(s) in arginine biosynthesis. These strains were designated phenotypically as Arg(CHG) according to the Salmonella typhimurium classification scheme. Staphylococcus simulans strains appeared to be either ArgA or Arg I. The ArgI strains of S. simulans and S. capitis had moderate to high ornithine carbamoyltransferase (EC 2.1.3.3) activities and therefore could not be designated as argI mutants. ArgI strains in other species had no or very low ornithine carbamoyltransferase activities. All of the natural Staphylococcus auxotrophs tested grew in the presence of L-citrulline and had moderate to high argininosuccinase (EC 4.3.2.1) activities. Arginine auxotrophs of species with a wide host range were often capable of reverting to arginine-independent or complete prototrophic growth, whereas auxotrophs of species that tended to be niche-specific and/or host-specific were incapable of reversion to arginine-independence, even in the presence of various mutagens. A relationship between the nature of arginine auxotrophy and habitat is suggested.     

Evidence for cooperation between cells during sporulation of the yeast Saccharomyces cerevisiae.

Diploid Saccharomyces cerevisiae cells heterozygous for the mating type locus (MATa/MAT alpha) undergo meiosis and sporulation when starved for nitrogen in the presence of a poor carbon source such as potassium acetate. Diploid yeast adenine auxotrophs sporulated well at high cell density (10(7) cells per ml) under these conditions but failed to differentiate at low cell density (10(5) cells per ml). The conditional sporulation-deficient phenotype of adenine auxotrophs could be complemented by wild-type yeast cells, by medium from cultures that sporulate at high cell density, or by exogenously added adenine (or hypoxanthine with some mutants). Adenine and hypoxanthine in addition to guanine, adenosine, and numerous nucleotides were secreted into the medium, each in its unique temporal pattern, by sporulating auxotrophic and prototrophic yeast strains. The major source of these compounds was degradation of RNA. The data indicated that differentiating yeast cells cooperate during sporulation in maintaining sufficiently high concentrations of extracellular purines which are absolutely required for sporulation of adenine auxotrophs. Yeast prototrophs, which also sporulated less efficiently at low cell density (10(3) cells per ml), reutilized secreted purines in preference to de novo-made purine nucleotides whose synthesis was in fact inhibited during sporulation at high cell density. Adenine enhanced sporulation of yeast prototrophs at low cell density. The behavior of adenine auxotrophs bearing additional mutations in purine salvage pathway genes (ade apt1, ade aah1 apt1, ade hpt1) supports a model in which secretion of degradation products, uptake, and reutilization of these products is a signal between cells synchronizing the sporulation process.     

A Cytological Analysis of the Antimetabolite Activity of 5-Hydroxyuracil in Vicia faba Roots

The effects of 5-hydroxyuracil (5-HU) (isobarbituric acid) upon cell elongation, mitosis, and DNA synthesis were studied in Vicia faba roots. 5-HU had no consistent effect upon root elongation. It blocked DNA synthesis (analyzed by photometric measurements of Feulgen dye in nuclei) during the first 6 hours of treatment; the block spontaneously disappeared by the 12th hour of treatment. Uracil and thymine had no effect upon this block of synthesis. Both thymidine and uridine reversed the block in 6 and 9 hours respectively. In all cases blockage of DNA synthesis was followed by inhibition of mitosis (determined by changes in the percentage of cells in mitosis) and resumption of DNA synthesis was followed by resumption of mitosis. Inhibition indices calculated from the mitotic data indicated a competitive relationship between 5-HU and thymidine and 5-HU and uridine. 5-HU is considered to block DNA synthesis by competing with thymidine for sites on enzymes involved in the synthesis. It is suggested that uridine reverses the block in synthesis by undergoing a conversion to thymidine.     

Occurrence of diploid strains of Cryptococcus neoformans.

A mating between niacin and pantothenate auxotrophs of Cryptococcus neoformans gave a few prototrophic progeny that were self-fertile. These were uninuclear but contained twice as much DNA as the parental strains. Segregation of nutritional markers was observed upon sporulation. We conclude that these self-fertile strains are diploids.     

Standard YPD, even supplemented with extra nutrients, does not always compensate growth defects of Saccharomyces cerevisiae auxotrophic strains

Conventional complex media are routinely used to grow auxotrophic strains under the assumption that they can compensate the latter's nutritional deficiencies. We here demonstrate that this is not always true. This study compares the growth parameters of Saccharomyces cerevisiae (S288C) and its derived auxotrophic strains FY1679-14C and BY4741 in synthetic minimal medium (SD), standard YPD medium from two of the most commonly used suppliers, or modified YPD medium. Maximum specific growth rates of auxotrophic strains were slightly lower than the prototrophic case in all growth conditions tested. Also, the biomass production of auxotrophic strains in synthetic medium was slightly less than the prototrophic case. However in both of the two standard YPD media used, the biomass production of both auxotrophic strains was markedly lower than that of the prototrophic one. The extent of the differences depended on the medium used. Indeed in one of the two YPD media, the lower biomass production of auxotrophic strains was evident even at the diauxic shift. Uracil seems to be the main limiting growth factor for both auxotrophic strains growing in the two standard YPD medium tested. No YPD media or specific supplement was able to compensate for the effect of the auxotrophic mutations in the multiple auxotrophic marker strain BY4741. The fact that auxotrophic strains grew poorly on YPD when compared to their prototrophic counterpart indicates that standard YPD medium is not sufficient to overcome the effect of auxotrophic mutations.     

Effects of growth temperatures on plating efficiencies and stabilities of heterokaryons of Candida albicans

Heterokaryons (hets) of Candida albicans constructed by fusing protoplasts of complementing auxotrophs produce heterogeneous clones on minimal medium consisting of (i) a minority of slow-growing hets, (ii) a preponderance of non-growing, parental-type auxotrophic monokaryons, and (iii) some prototrophic monokaryons bearing hybrid nuclei. Hets grown at a given temperature within the range 25° C to 41° C replate with higher efficiencies at any lower temperature and exhibit progressively declining plating efficiencies as plate temperatures increase beyond that at which they were initially grown. Neither auxotrophic nor prototrophic monokaryons show such responses. Growth of colonies produced by hets, wild-type strains or prototrophic hybrid monokaryons is stimulated by temperatures in the order, 37° C > 30° C > 41° C > 25° C. However, the proportion of hets to auxotrophic monokaryons within individual net clones increases directly from 25° C to 41° C. Though this pattern obtains whether colonies are compared at equivalent sizes or ages, het frequencies decline as colonies age at all temperatures. Appearance of hybrid monokaryons within het clones is unaffected by growth temperature. The relationships of temperatures to plating efficiencies and stabilities of hets are independent of the natures of their complementing auxotrophies or the wild-type backgrounds of their nuclear components and are, therefore, functions of heterokaryosis per se. Modifications of these relationships by selective metabolic antagonists or by growth of hets on different preand post-plating carbon sources indicate that they reflect temperature-dependent properties of mitochondria which are peculiar to hets.     

Changes in macromolecular synthesis in Xanthomonas oryzae infected with bacteriophage XP-12.

Phage XP-12, which has complete substitution of the cytosine residues in its DNA with 5-methylcytosine residues, was shown to inhibit incorporation of uracil into host DNA and RNA during the latent period. This apparent inhibition of host macromolecular synthesis was not accompanied by extensive degradation of the host chromosome. Phage DNA synthesis in infected cells occurred at a faster rate than host DNA synthesis in analogous uninfected cells. However, phage DNA synthesis could not be accurately monitored by incorporation of [methyl-3H]thymidine into DNA because, soon after infection, there was a marked inhibition of utilization of exogenous thymidine for DNA synthesis. Phage infection conferred upon a thymine auxotrophic host the ability to synthesize thymine nucleotides for phage DNA synthesis. It is suggested that a phage-induced thymidylate synthetase activity is partially responsible for the inhibition of thymidine incorporation.