Ultraviolet photoproducts at the ochre suppressor mutation site in the glnU gene of Escherichia coli: relevance to "mutation frequency decline"

Summary Ochre suppressor mutations induced by UV in the Escherichia coli glnU tRNA gene are CG to TA transitions at the first letter of the anticodon-encoding triplet, CAA. Premutational UV photoproducts at this site have long been known to exhibit an excision repair anomaly (mutation frequency decline or MFD), whereby post-irradiation inhibition of protein synthesis enhances their excision and reduces suppressor mutation yields ten-fold. We sought to clarify the basis of this unique repair response by determining the spectrum of UV photoproducts on both strands of a 36 by region of glnU which includes the anticodon-encoding triplet. We found that four different photolesions are produced within the 3 by sequence corresponding to the tRNA anticodon: (i) on the transcribed strand, TC (6–4) photoproducts and TC cyclobutane dimers are formed in equal numbers at the site of the C to T transition, indicating that this site is a hotspot for the usually less frequent (6–4) photoproduct; (ii) on the nontranscribed strand, TT dimers are found opposite the second and third letters of the anticodon-encoding triplet, adjacent to the mutation site; and (iii) on the nontranscribed strand, an alkali-sensitive lesion other than a (6–4) photoproduct is formed, apparently at the G in the mutation site. We suggest that mutation frequency decline may reflect excision repair activity at closely spaced UV lesions on opposite strands, resulting in double-strand breaks and the death of potential mutants.     

Regulation of pyrimidine biosynthesis in Pseudomonas cepacia

Pyrimidine biosynthesis was investigated in Pseudomonas cepacia ATCC 17759. The presence of the de novo pyrimidine biosynthetic pathway enzyme activities was confirmed in this strain. Following transposon mutagenesis of the wild-type cells, a mutant strain deficient for orotidine 5-monophosphate decarboxylase activity (pyrF) was isolated. Uracil, cytosine or uridine supported the growth of this mutant. Uracil addition to minimal medium cultures of the wild-type strain diminished the levels of the de novo pyrimidine biosynthetic enzyme activities, while pyrimidine limitation of the mutant cells increased those de novo enzyme activities measured. It was concluded that regulation of pyrimidine biosynthesis at the lelel of enzyme synthesis in P. cepacia was present. Aspartate transcarbamoylase activity was found to be regulated in the wild-type cells. Its activity was shown to be controlled in vitro by inorganic pyrophosphate, adenosine 5-triphosphate and uridine 5-phosphate.     

Prebiotic synthesis of diaminopyrimidine and thiocytosine

The reaction of guanidine hydrochloride with cyanoacetaldehyde gives high yields (40–85%) of 2,4-diaminopyrimidine under the concentrated conditions of a drying lagoon model of prebiotic synthesis, in contrast to the low yields previously obtained under more dilute conditions. The prebiotic source of cyanoacetaldehyde, cyanoacetylene, is produced from electric discharges under reducing conditions. The effect of pH and concentration of guanidine hydrochloride on the rate of synthesis and yield of diaminopyrimidine were investigated, as well as the hydrolysis of diaminopyrimidine to cytosine, isocytosine, and uracil. Thiourea also reacts with cyanoacetaldehyde to give 2-thiocytosine, but the pyrimidine yields are much lower than with guanidine hydrochloride or urea. Thiocytosine hydrolyzes to thiouracil and cytosine and then to uracil. This synthesis would have been a significant prebiotic source of 2-thiopyrimidines and 5-substituted derivatives of thiouracil, many of which occur in tRNA. The applicability of these results to the drying lagoon model of prebiotic synthesis was tested by dry-down experiments where dilute solutions of cyanoacetaldehyde, guanidine hydrochloride, and 0.5m NaCl were evaporated over varying periods of time. The yields of diaminopyrimidine varied from 1 to 7%. These results show that drying lagoons and beaches may have been major sites of prebiotic syntheses.     

Use of synthetic lethal mutants to clone and characterize a novel CTP synthetase gene in Saccharomyces cerevisiae

In the pyrimidine biosynthetic pathway, CTP synthetase catalyses the conversion of uridine 5-triphosphate (UTP) to cytidine 5-triphosphate (CTP). In the yeast Saccharomyces cerevisiae, the URA7 gene encoding this enzyme was previously shown to be nonessential for cell viability. The present paper describes the selection of synthetic lethal mutants in the CTP biosynthetic pathway that led us to clone a second gene, named URA8, which also encodes a CTP synthetase. Comparison of the predicted amino acid sequences of the products of URA7 and URA8 shows 78% identity. Deletion of the URA8 gene is viable in a haploid strain but simultaneous presence of null alleles both URA7 and URA8 is lethal. Based on the codon bias values for the two genes and the intracellular concentrations of CTP in strains deleted for one of the two genes, relative to the wild-type level, URA7 appears to be the major gene for CTP biosynthesis. Nevertheless, URA8 alone also allows yeast growth, at least under standard laboratory conditions.     

Ozonolysis of Thymidine: Isolation and Identification of the Main Oxidation Products

The ozone-mediated oxidation of thymidine was investigated on the basis of final product identification. The oxidation reaction gave rise to five major modified nucleosides which were isolated and characterised from extensive H NMR and mass spectrometry studies. The comparison with the current knowledge of the hydroxyl radical-mediated oxidation reactions of thymidine in aerated aqueous solution indicates that the formation of ozone oxidation products may be mostly explained in terms of initial generation of an ozonide. Indeed, the identified products obtained by ozonolysis of thymidine resulted from the opening of the pyrimidine C5-C5 bond.     

Mobilization of intracellular calcium in cultured vascular smooth muscle cells by uridine triphosphate and the calcium ionophore A23187

The known action of uridine triphosphate (UTP) to contract some types of vascular smooth muscle, and the present finding that it is more potent than adenosine triphosphate in eliciting an increase in cytosolic Ca²⁺ concentration in aortic smooth muscle, led us to investigate the mode of action of this nucleotide. With this aim, cultured bovine aorta cells were subjected to patch-clamp methodologies under various conditions. Nucleotide-induced variations in cytosolic Ca²⁺ were monitored by using single channel recordings of the high conductance Ca²⁺-activated K⁺ (Maxi-K) channel within on-cell patches as a reporter, and whole-cell currents were measured following perforation of the patch. In cells bathed in Na⁺-saline, UTP (>30 nm) induced an inward current, and both Maxi-K channel activity and unitary current amplitude of the Maxi-K channel transiently increased. Repetitive exposures elicited similar responses when 5 to 10 min wash intervals were allowed between challenges of nucleotide. Oscillations in channel activity, but not oscillation in current amplitude were frequently observed with UTP levels > 0.1 m. Cells bathed in K⁺ saline (150 m) were less sensitive to UTP (5-fold), and did not show an increase in unitary Maxi-K current amplitude. Since the increase in amplitude occurs due to depolarization of the cell membrane, a change in amplitude was not observed in cells previously depolarized with K⁺ saline. The enhancement of Maxi-K channel activity in the presence of UTP was not diminished by Ca²⁺ entry blockers or by removal of extracellular Ca²⁺. However, in the latter case, repetitive responses progressively declined. These observations, as well as data comparing the action of low concentrations of Ca²⁺ ionophores (<5 m) to that of UTP indicate that both agents elevate cytosolic Ca²⁺ by mobilization of this ion from intracellular pools. However, the Ca²⁺ ionophore did not cause membrane depolarization, and thus did not change unitary current amplitude. The effect of UTP on Maxi-K channel activity and current amplitude was blocked by pertussis toxin and by phorbol 12-myristate 13-acetate (PMA), but was not modified by okadaic acid, or by inhibitors of protein kinase C (PKC). Our data support a model in which a pyrimidinergic receptor is coupled to a G protein, and this interaction mediates release of Ca²⁺ from intracellular pools, presumably via the phosphatidyl inositol pathway. This also results in activation of membrane channels that give rise to an inward current and depolarization. Ultimately, smooth muscle contraction ensues. PKC does not appear to be directly involved, even though the UTP response is blocked by low nm levels of PMA. While the latter data implicate PKC in diminishing the UTP response, agents that inhibit either PKC or phosphatase activity did not prevent abolition of UTP responses by PMA, nor did they modify basal channel activity.     

The possible role of soluble salts in chemical evolution

Summary A model is proposed for a prebiotic environment in which concentration, condensation, and chemical evolution of biomolecules could have taken place. The main reactions expected of proteins, nucleic acids, lipids, and some of their precursors in this environment are examined.The model is based on our previously developed concept of a fluctuating system in which hydration and dehydration processes take place in a cyclic manner. In the present model, however, high concentrations of soluble salts, such as chlorides and sulfates, are taken into account, whereas previously a more or less salt-free system had been assumed. Thus the preponderance of surfaces of soluble salts is implied, even though sparingly soluble minerals, such as clay minerals or quartz, are also present.During the dehydration stage biomolecules tend to leave the solution and concentrate at certain microenvironments, such as in micelles and aggregates, at the liquid-gas surface and, possibly, at the emerging solid surfaces. Moreover, in these brines, and especially during the last stages of dehydration, high temperatures are attainable, which may enhance certain reactions between the organic molecules, and result in a net increase of condensation over degradation.In the dehydrated state, solid-state condensation and synthesis reactions are possible in which the surface of soluble salts may serve as a catalyst. Several reports in the literature support this hypothesis. Hydration brings about dissolution of the minerals and redistribution of the biomolecules. In such a system, evolutionary processes like those postulated by White (1980) and by Lahav and White (1980) are possible. Moreover, since several soluble salts of known geological occurrence are optically active in their crystalline state, the involvement of the model system in the selection and evolution of chiral organic compounds should also be considered. In addition, organic molecules in the above microenvironments are also expected to undergo selective interactions based on factors such as molecular pattern and chiral recognition and hydrophobicity. The proposed system emphasizes the need to develop the theoretical background and experimental methods for the study of interactions among biomolecules in the presence of high salt concentrations and solid surfaces of soluble salts, as well as interactions between the biomolecules and these surfaces.     

Degradation of pyrimidine bases in Clostridium sticklandii

Resting cells of Clostridium sticklandii took up thymine or uracil, when grown in a medium containing 40 mM serine and 20 mM thymine or uracil. The uptake was much lower, when the cells had been grown in a complex medium. Cell-free extracts from cells grown in the complex medium reduced the two bases to the dihydro compounds and decomposed dihydrothymine to -ureidoisobutyrate, as indicated by thin-layer chromatography. Uptake and degradation were stimulated by both NADH and NADPH. Further breakdown did not occur, as ¹⁴CO₂ was not evolved from C-2-labelled thymine or uracil. The rates of pyrimidine uptake and breakdown of C. sticklandii were lower than those reported for C. sporogenes (Hilton et al., 1975).     

Purification and Characterization op a 31-Kilodalton Iron-Regulated Periplasmic Protein from Pasteurella Haenolytica A1

ABSTRACT

A prominent iron-regulated periplasmic protein was purified from Pasteurella haemolytica grown in an iron-deficient chemically defined medium. The protein was purified by anion exchange chromatography and appeared as a single band by SDS-PAGE with a molecular weight of 32,000. A yield of five mg was obtained from 91 mg of protein extract. The iron-regulated protein existed as a monomer in the native state with an average molecular weight of 29,877 as determined by analytical ultracentrifugation. The protein had a molecular weight of 30,880 as determined by matrix-assisted laser desorption mass spectrometry, hence the protein is referred to as the 31 kDa protein. Isoelectric focusing showed four bands with pIs of 7.15, 6.8, 6.6, and 5.9, The secondary structure of the protein was determined by circular dichroism and contained 16% α-helical structure. The N-terminal sequence, EPFKVVTTFTVIQDIAQNVAGDKAT, showed a 95% identity with the 31 kDa iron-binding protein from Haemophilus influenzae. Isolation and characterization of iron-regulated proteins are of particular interest because of their potential roles in iron assimilation and microbial virulence.     

Pyrimidine and Purine Analogues,Effects on Cell Cycle Regulation and the Role of Cell Cycle Inhibitors to Enhance Their Cytotoxicity

In anti-cancer treatment, deoxynucleoside analogues are widely used in combination chemotherapy. Improvement can be achieved by rational design of novel combinations with cell cycle inhibitors. These compounds inhibit protein kinases, preventing the cell cycle from continuing when affected by deoxynucleoside analogs. The efficacy is dependent on the site of cell cycle inhibition, whether multiple cyclin-dependent kinases are inhibited and whether the inhibitors should be given before or after the deoxynucleoside analogs. The action of cell cycle inhibition in vivo may be limited by unfavorable pharmacokinetics. Preclinical and clinical studies will be discussed, aiming to design improved future strategies.