Modulation of restriction enzyme-induced damage by chemicals that interfere with cellular responses to DNA damage: a cytogenetic and pulsed-field gel analysis

The electroporation of restriction enzymes into mammalian cells results in DNA double-strand breaks that can lead to chromosome aberrations. Four chemicals known to interfere with cellular responses to DNA damage were investigated for their effects on chromosome aberrations induced by AluI and Sau3AI; in addition, the number of DNA double-strand breaks at various times after enzyme treatment was determined by pulsed-field gel electrophoresis (PFGE). The poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide (3AB) dramatically increased the yield of exchanges and deletions and caused a small but transitory increase in the yield of double-strand breaks induced by the enzymes. 1-beta-D-Arabinofuranosylcytosine, which can inhibit DNA repair either by direct action on DNA polymerases alpha and delta or by incorporation into DNA, potentiated aberration induction but to a lesser extent than 3AB and did not affect the amount of DNA double-strand breakage. Aphidicolin, which inhibits polymerases alpha and delta, had no effect on AluI-induced aberrations but did increase the aberration yield induced by Sau3AI. The postreplication repair inhibitor caffeine had no effect on aberration yields induced by either enzyme. Neither aphidicolin nor caffeine modulated the amount of DNA double-strand breakage as measured by PFGE. These data implicate poly(ADP-ribosyl)ation and polymerases alpha and delta as important components of the cellular processes required for the normal repair of DNA double-strand breaks with blunt or cohesive ends. Comparison of these data with the effect of inhibitors on the frequency of X-ray-induced aberrations leads us to the conclusion that X-ray-induced aberrations can result from the misjoining or nonrejoining of double-strand breaks, particularly breaks with cohesive ends, but that this process accounts for only a portion of the induced aberrations.     

Mutations induced by ionizing radiation in a plasmid replicated in human cells. I. Similar, nonrandom distribution of mutations in unirradiated and X-irradiated DNA

The Escherichia coli supF gene encoding the suppressor tyrosine tRNA in a human shuttle plasmid, pZ189, was used as a target for molecular analysis of X-ray-induced mutations in human lymphoblastoid cells. Following replication of the in vitro-irradiated plasmid in human cells, the mutant supF-containing molecules were cloned by phenotypic screening in E. coli and the nature of the mutations was determined by direct sequencing of the tRNA gene. At 160 Gy the mutant frequency was 13 times (0.39%) that observed in unirradiated controls (0.031%). When control plasmid was replicated directly in E. coli, the mutant frequency was 16 times less than that of the plasmid passaged through the human cells. The distribution of mutations was highly nonrandom and remarkably similar in both irradiated and control DNAs. The majority of the mutations were transitions involving G.C pairs and occurred selectively at most 5'-TC (3'-AG) sequences. These mutations at C's were preferentially distributed in the nontranscribed strand. We propose that mutations in the control plasmid result from oxidative damages that occur during and/or after its incorporation into human cells and that these damages are similar to those induced by ionizing radiation. The hot spots for mutations suggest that the proximate nucleotide sequence and the overall conformation of the target DNA are important in the production and/or processing of these damages during repair and replication.     

ECOLOGICAL CONSEQUENCES AND PHENOTYPIC CORRELATES OF PETAL SIZE VARIATION IN WILD RADISH,RAPHANUS SATIVUS (BRASSICACEAE)

In this paper we examine some ecological consequences and phenotypic correlates of flower size variation in wild radish, Raphanus sativus. Mean corolla diameter varied significantly among individuals within natural populations of R. sativus in California. On the average, almost 40% of flower biomass was allocated to corolla tissue. In field experiments, pollinator visitation increased significantly with corolla size. Large flowers also accumulated more nectar when pollinators were excluded from plants. In three populations, corolla size was positively correlated with allocation to pollen per flower (either anther weight or pollen grain number), but there was usually no phenotypic relationship between corolla size and several measures of female allocation (ovule number per flower, proportion fruit set, and total seed mass per fruit). Plants growing in the field produced fewer large flowers per unit of stem, and stem biomass was negatively related to corolla size for plants grown under controlled greenhouse conditions. Male and female fitness may covary differently with allocation to attractive floral features in species such as R. sativus, where seed production is often limited by resources rather than by pollen.     

Concentration dependence of proteoglycan diffusion

The mutual diffusion coefficient of the bovine nasal cartilage proteoglycan subunit is found to increase rapidly with increasing concentration and decreasing ionic strength. These results have been obtained by analysis of the boundary relaxation of concentration gradients in the analytical ultracentrifuge by schlieren optics. The diffusion behavior can be understood in terms of the nonideality of the proteoglycan. The magnitude of the nonideality is dominated by charge interactions, whereas the influence of molecular size and associated excluded-volume interactions is small. The concentration dependence of the apparent diffusion coefficient of the proteoglycan subunit from dynamic light scattering was found, in contrast, to decrease with increasing concentration. Computer simulation of the dynamic light scattering suggests that the presence of a small population of aggregates may account for the difference in the two types of diffusion measurement due to their marked influence on the scattering.     

Patterns of dental wear in the Lengua Indians of Paraguay

Wear patterns were examined on dental casts of 202 living Lengua Indians from the Chaco area of Paraguay. Consideration was given to the development of the molar helicoidal plane, age-related changes in occlusal attrition, coalescence of dentine exposures, interproximal attrition, and erupted crown height. This study lends support to Osborn's theory of the helicoidal plane development by showing that attrition enhances rather than modifies posteruption molar occlusal planes. The rate of interproximal attrition was found to slow down with the eruption and functional initiation of the third molars. Sinuous and cavo-convex interproximal contact areas that are generated with age, however, appeared to be less abrasion resistant than straight surfaces, hence leading to an increase in interproximal attrition rates with advanced age. Maximum crown height reduction occurred between the ages of 20 and 40 years in central incisors, canines, and first molars. Kruskal-Wallis tests and log linera models failed to demonstrate significant sexually dimorphic or antimeric differences in wear patterns of Lengua teeth.     

The S-state dependence of Cl binding to plant Photosystem II

A combined single-turnover flash and ³⁵Cl NMR technique has been used to monitor S-state dependence of Cl⁻ binding to PS-II particles derived from mangrove (Avicennia marina). No detectable high-affinity binding was found to particles in the S₀ and S₁ states, but binding with an affinity comparable to that which activates O₂ evolution was found in the S₂ and S₃ states.     

Glycosaminoglycan synthesis is depressed during mitosis and elevated during early G1

[35S]Sulfate incorporation was measured in populations of Chinese hamster ovary cells enriched for mitotics, early G1 cells, and interphase monolayers or suspensions. Incorporation was determined by biochemical analysis of extracts and quantitative autoradiography of thick sections. 90% of [35S]sulfate was incorporated into glycosaminoglycan (GAG). Incorporation was depressed fourfold in mitotics and stimulated by from two- to three-fold in early G1 cells relative to mixed interphase cells. GAG synthesis was maintained into late G2. Thus, the rate of GAG biosynthesis was correlated temporally with the detachment and reattachment of cells to substrate. Inhibitors of protein synthesis brought about the rapid arrest of GAG biosynthesis. However, xylosides, which bypass the requirement for core protein, did not bring oligosaccharide sulfation in mitotics to interphase levels. These observations indicate an inhibition of Golgi processing and are consistent with a generalized defect of membrane vesicle-mediated transport during mitosis.     

Amoeboid locomotion of Acanthamoeba castellanii with special reference to cell-substratum interactions

The amoeboid locomotion of Acanthamoeba castellanii has been studied by observation of individual cells moving on a planar glass substratum. Cell-substratum interactions involved in traction have been observed by reflexion interference microscopy. A variable part of the ventral surface of A. castellanii formed a protean platform, the 'associated contact', from which filopodia were subtended; these established stable, focal adhesions (approximately 0.4 micron diameter) on the substratum beneath. Surprisingly, acanthopodia, a prominent feature of this protozoon, did not play an obvious role in traction. The dimensions of the cell-substratum gap in the associated contact could be modulated by the concentration of ambient electrolyte. Dilution of electrolyte from 50 mM-KC1 to 2mM resulted in (i) an increase in the cell-substratum gap, (ii) a marked decrease in cell motility, (iii) reduced cell adhesion to glass.     

Adrenalectomy of Rats Results in Hypomyelination of the Central Nervous System

The effect of adrenalectomy on CNS myelin accumulation was investigated to determine whether glucocorticoids play a role in regulating myelination. When 14-day-old rats were adrenalectomized and sacrificed 7-8 days later, the amount of bulk-isolated myelin in whole brain, as expressed per gram wet weight of brain or per milligram DNA-phosphate, was reduced to about 75% that of sham-operated controls. Both brain weight and DNA content were unchanged by adrenalectomy. Examination of individual brain regions also revealed decreased amounts of myelin in adrenalectomized animals. Brain glycerol 3-phosphate dehydrogenase specific activity was reduced in adrenalectomized animals to 40-60% that of controls, and serum corticosterone levels were less than 0.6% of control levels. The amount of cerebral myelin in animals adrenalectomized on day 21 and sacrificed 9 days later was not significantly reduced. This suggests a possible role of glucocorticoids during the early period of rapid myelination.