Quantitation and Visualization of Ultraviolet‐Induced DNA Damage Using Specific Antibodies: Application to Pigment Cell Biology

The major types of DNA damage induced by sunlight in the skin are DNA photoproducts, such as cyclobutane pyrimidine dimers (CPDs), (6‐4)photoproducts (6‐4PPs) and Dewar isomers of 6‐4PPs. A sensitive method for quantitating and visualizing each type of DNA photoproduct induced by biologically relevant doses of ultraviolet (UV) or sunlight is essential to characterize DNA photoproducts and their biological effects. We have established monoclonal antibodies specific for CPDs, 6‐4PPs or Dewar isomers. Those antibodies allow one to quantitate photoproducts in DNA purified from cultured cells or from the skin epidermis using an enzyme‐linked immunosorbent assay. One can also use those specific antibodies with in situ laser cytometry to visualize and measure DNA photoproducts in cultured cells or in the skin, using indirect immunofluorescence and a laser‐scanning confocal microscope. This latter method allows us to reconstruct three‐dimensional images of nuclei containing DNA photoproducts and to simultaneously examine DNA photoproducts and histology in multilayered epidermis. Using those techniques, one can determine the induction and repair of these three distinct types of DNA photoproducts in cultured cells and in the skin exposed to sublethal or suberythematous doses of UV or solar simulated radiation. As examples of the utility of these techniques and antibodies, we describe the DNA repair kinetics following irradiation of human cell nuclei and the photoprotective effect of melanin against DNA photoproducts in cultured pigmented cells and in human epidermis.     

A seed parasitoid wasp prevents berries from changing their colour,reducing their attractiveness to frugivorous birds

1. Frugivorous and seed‐feeding insects may alter the traits of fruits, such as shape and size, which may influence fruit attractiveness to frugivorous birds. Consequently, trait‐mediated interactions may occur in systems where plants, seed‐dispersing frugivorous vertebrates, and frugivorous or seed‐feeding insects interact. We investigated colour manipulation in Ilex integra Thunb. berries caused by the seed parasitoid wasp Macrodasyceras hirsutum Kamijo and how that manipulation relates to fruit attractiveness for frugivorous birds. 2. In winter, the colour of I. integra berries varied from green to red, but most berries were greenish, indicating that the berries were immature. Berry dissection indicated that the number of live parasitoid larvae present within each berry was closely related to berry colour – the greater the number of live larvae, more intense is the green colour of the berry. However, the wasp larvae did not modify the shape or size of the berries. More than 98% of berries that were protected from the insects by gauze bags ripened and turned red. In the present study, berries with unfertilised seeds alone turned red. Field‐feeding preference tests showed that the brown‐eared bulbul Hypsipetes amaurotis Temminck preferred red berries to green berries. 3. We demonstrated that the seed parasitoid wasp manipulates the berry colour, but not its shape or size, in a density‐dependent manner. Because green berries suffered less from bird foraging, we believe that this colour manipulation helps the wasps to avoid being killed by the birds. The present study indicates that manipulation by wasps may reduce the level of mutualism between the tree and seed‐dispersing birds.     

INSTABILITY OF MESSENGER AND RIBOSOMAL RNA IN A GLUE-PROTEIN MUTANT OF Bombyx mori

The mucous glands of Bombyx pupae secrete glue proteins which attach deposited eggs to the mounting sheet. A mutant of a dominant gene, named no glue (Ng) , produces nonadhesive eggs which have a low capacity for glue-protein synthesis. In the present study it was shown that the mucous glands of Ng silkworms showed rapid degradation of mRNA as well as rRNA during development; this may cause the low capacity for glue-protein synthesis in the mutant organ. In contrast, the mucous glands of normal silkworms showed a significant increase in content of RNA's until the maximum rate of glue-protein synthesis was achieved. The degradation of RNA in the Ng mucous gland was inhibited by actinomycin D injected into the body fluid. Thus it is supposed that the Ng gene codes for a presumptive controller RNA, which would be the mediator of RNA instability in the mucous glands of Ng pupae.     

Translation of RNA from Eggs During Post-Diapause Development of Bombyx mori

Eggs of Bombyx mori are aroused from diapause by long-term chilling and develop when transferred to 25°C. During the first 20 hr of post-diapause development, the polysome content and the presumed rate of protein synthesis increase about 3-fold, while the ribosome content and the total RNA content increase only 1.1-fold. In this study, total RNAs were extracted from chilled eggs (termed 0 hr of development), and post-diapause eggs at 10 and 20 hr of development. The RNAs were purified further by high pressure liquid chromatography to remove RNA-like oligonucleotides. On translation in a protein-synthesizing system derived from wheat germ with a subsaturating amount of RNA, no difference was found in the relative amounts of translatable mRNA activity at 10 and 20 hr of development from that at 0 hr. Moreover, the translation products of the different RNA preparations in a rabbit reticulocyte lysate system appeared very similar when separated by gel electrophoresis and located by fluorography. These facts suggest that protein synthesis in early post-diapause development is controlled at a translational level.     

Effect of blue light on the photonastic reaction of rice leaves

Effect of blue light on the epinastic reaction of rice plantleaves (Oryza saliva L.) was studied. The inclination angleof intact leaves, when the plants were grown either in the lightor in the dark, was greatly increased by blue light irradiationbut not much increased by green, yellow, orange, red or whitelight. The effect of blue light on the epinastic reaction was,however, obscure in excised leaves, especially when they wereplaced on the horizontal. The photonastic reaction requireda prolonged irradiation of high intensity. The photosensitivesite localized at the leaf-nodal part. Response spectra forthe photonastic reaction were characterized by 2 distinct peaksat 420–440 mµ and 460–480 mµ and a fairlystrong response in the near ultra-violet region. (Received April 17, 1969; )     

The Relationship of Some Glycosidases to the Endogenous and IAA-induced Growth of Light-grown Cucumber Hypocotyls

Some glycosidases in light-grown cucumber (Cucumis sativus L. cv. Aonaga-jibae) hypocotyl sections were examined with respect to their localization and relation to endogenous and IAA-induced growth. Frozen-thawed sections were used directly for measurement of enzyme activities, and β-glucosidase, α- and β-galactosidases and β-xylosidase were assayed by using p- or o-nitro-phenylglycopyranosides as substrates. The order of the activity of these enzymes were β -glucosidase > β -galactosidase =α-galactosidase > β-xylosidase. No activity of α-glucosidase was detected. High glycosidase activities were found in the youngest region of the hypocotyl, where the endogenous growth rate was highest. However, there was no significant difference in the activities of this region between seedlings at different growth stages. Among the enzymes tested, β -glucosidase showed a high correlation with the endogenous growth rate. β-glucosidase was found to be mostly associated with the cell wall fraction, while β-galactosidase was rather found in the soluble fraction of the cell. Separation of the epidermis from the section showed that a very high activity of β-glucosidase was associated with the epidermis. In both whole sections and isolated cell wall fractions, IAA was shown to have no effect on the activities of β-glucosidase and β-galactosidase.     

Homoeotic Gene ETc Controls the Cell Number of Embryonic Ectodermal Layers of Bombyx mori

The formation of segments of Bombyx larvae involves differentiation of legged and legless segments during embryogenesis. Observations by light microscopy of serial sections of developing embryos of Bombyx showed that the cell number of the ectodermal layers increased more rapidly in segments where legs were being extruded than in those where no appendages were formed. In the embryos of a homoeotic mutant for the E -pseudoallelic locus (about 0.0–VI), E^Tc/E^Tc , in which all the abdominal segments were legless, the cell number of the ectodermal layers did not increase as in normal embryos. These findings suggest that the E^Tc gene controls the cell number of the ectodermal layers in relation to the differentiation of abdominal segments.     

Initiation of Sperm Motility Induced by Cyclic AMP in Hamster and Boar

When the plasma membrane of hamster and boar spermatozoa was extraced by treatment with Triton X-100 and the demembranated spermatozoa were transferred to a reactivating medium containing only ATP, axonemes were initially immotile, and then gradually became motile. Under these experimental conditions, the cAMP content in the reactivating medium increased soon. This suggests that cAMP is synthesized from ATP by adenylate cyclase involved in incompletely removed or solubilized residual sperm membrane and that the autosynthesized cAMP causes the delay in motility initiation. This delayed initiation of motility did not occur when phosphodiesterase was added to the reactivating medium and the phosphodiesterase-dependent quiescent sperm became motile instantaneously at any time when excess cAMP was supplemented. Furthermore, demembranated sperm which were diluted in the reactivating medium containing ATP and cAMP, immediately became motile. cAMP levels in the cell increased during the initiation of sperm motility in both species. These results suggest that cAMP is the real factor indispensable for the initiation of sperm motility at ejaculation in mammals.     

Cell Fusion and Its Application to Genetic Analysis of Development in Dictyostelium discoideum

Dictyostelium cells were fused by a modification of the polyethylene glycol method of Kuhn and Parish. In the modified method Tricine buffer and Concanavalin A were used in place of Ca⁺⁺. The efficiency of genetic complementation through cell fusion was about 10 times higer by the modified method than by the original method with glycine buffer and Ca⁺⁺. Complementation between developmental mutants without any selectable growth character was clearly detected by the modified system, at efficiencies of about 1 in 10–20 surviving cells.