Klaus Kalthoff

ACTION SPECTRA FOR UV INDUCTION AND PHOTOREVERSAL OF A SWITCH IN THE DEVELOPMENTAL PROGRAM OF THE EGG OF AN INSECT (SMITTIA)

Abstract— An action spectrum was established for the induction by ultraviolet (UV) radiation of the aberrant body segment pattern ‘double abdomen’ in the egg of the Chironomid midge Smittia. The action spectrum shows a peak at 285 nm wavelength, a shoulder at 265 nm, a slight increase from 245 to 240 nm, and a steep decline towards 300 nm. Corrections for wavelength‐dependent shielding within the egg result in transformation of the shoulder at 265 nm into a minor peak. These results are compatible with the assumption that a nucleic acid‐protein complex is involved in the initial photoreaction. This assumption is supported by the fact that the UV induction of the aberrant body segment pattern ‘double abdomen’ in the egg of Smittia is photoreversible. Wavelengths effective in this photoreversal range from 310 to 460 nm, with a peak at 440 nm. These spectral characteristics agree with action spectra for photoreactivation in other systems. Indirect photoreactivation does not occur in the egg of Smittia under the conditions of the experiments. Photoreversal with 440 nm radiation at high dose rate is temperature‐dependent. The results support the assumption that the molecular basis for photoreversal in the egg of Smittia is similar to the predominant molecular mechanism of direct photoreactivation in other systems. Targets possibly involved in the UV induction of the ‘double abdomen’ are discussed.

PHOTOREACTIVATION OF RNA IN UV-IRRADIATED INSECT EGGS (SMITTIA SP., CHIRONOMIDAE, DIPTERA) I. PHOTOSENSITIZED PRODUCTION AND LIGHT-DEPENDENT DISAPPEARANCE OF PYRIMIDINE DIMERS*

Abstract. Irradiation of Smittia eggs with UV during intravitelline cleavage causes the formation of pyrimidine dimers in the (largely ribosomal) RNA of the eggs. The yield of dimers is wavelength‐dependent in a way that strongly suggests the involvement of photosensitizing egg components. Illumination of UV‐irradiated eggs with light (380 or 400 nm) causes both photoreactivation of the eggs and mono‐merization of the pyrimidine dimers in their RNA. The photoreactivable sector of the biological damage is correlated with the amount of pyrimidine dimers present in the RNA after inactivation of the eggs with UV of different wavelengths. The data are regarded as the first direct evidence that the photoreactivation of a eukaryotic organism is correlated with the light‐dependent (and apparently enzymatic) monomerization of pyrimidine dimers in RNA.

Photoreversion of UV induction of the malformation “double abdomen” in the egg of Smittia spec. (Diptera, Chironomidae)☆

The UV induction of the malformation “double abdomen” in the eggs of Smittia is photoreversible with a constant dose reduction factor of about 0.7 under certain experimental conditions. The qualitative effect was shown to be significant (p < 0.01) under various conditions. The near-UV and visible radiation employed for photoreversion did not cause any delay of development and was effective only after, not before, UV irradiation. The results are discussed with regard to direct or indirect photoreversion. From current knowledge about photoreversion in living systems, the results can be regarded as evidence that at least one effective target is a nucleic acid. Since photoreversible UV induction of double abdomens can be achieved by irradiation of a strictly cytoplasmic area, the results are consistent with the hypothesis that the factors which determine the metameric pattern in the eggs of Smittia include the activity of one or more ooplasmic nucleic acids.

PHOTOREACTIVATION OF RNA IN UV‐IRRADIATED INSECT EGGS (SMITTIA SP., CHIRONOMIDAE, DIPTERA) II. EVIDENCE FOR HETEROGENEOUS LIGHT‐DEPENDENT REPAIR ACTIVITIES*

Abstract. Two biological effects of UV radiation upon Smittia eggs are observed, both of which seem to be associated with the formation of pyrimidine dimers in the RNA (largely ribosomal) of the eggs. While irradiation of the anterior pole region causes the formation of an aberrant segment pattern (double abdomen induction), irradiation of entire eggs leads to an arrest of their development (inactiva‐tion). Both UV effects are photoreversible with different action spectra of the photoreactivating light. A dose rate dependence of the photoreactivation can be observed after both UV effects. The saturating dose rate is about 6 W/m2 (at 440 nm) after UV induction of double abdomens. Upon UV inactivation, the saturating dose rate level for the photoreactivating light is much higher, and a single light flash causes both a considerable biological reactivation and the disappearance of about 7 × 109 pyrimidine dimers from the total RNA per egg. The results indicate the presence of heterogeneous light‐dependent repair activities acting upon UV induced pyrimidine dimers in the RNA of the eggs.

RNA and protein synthesis in developing embryos ofSmittia spec. (Chironomidae, Diptera)

SummaryEmbryos of the chironomid midgeSmittia spec. were permeabilized with sodium hypochlorite and octane. Uptake of labeled uridine and amino acids suggested that these compounds are actively transported across the plasma membrane. Before blastoderm formation, uridine was incorporated at low levels into nuclear DNA and mitochondrial RNA. After blastoderm formation, uridine was incorporated rapidly, mostly into cytoplasmic RNA including both ribosomal RNA precursors and poly(A)-containing RNA. Protein synthesis was observed throughout early embryogenesis. By measuring incorporation of labeled amino acids and internal amino acid pool sizes, we found that the rate of protein synthesis increased with development. Experiments with inhibitors of transcription indicated that proteins synthesized before blastoderm formation were translated from maternal mRNA. During blastoderm stages, embryonic mRNAs seemed to replace maternal mRNAs. Proteins synthesized during short incubation periods in vivo were separated by two-dimensional gel electrophoresis. After blastoderm formation, several new proteins were found that could not be detected at earlier stages.

Inhibition by ultraviolet light of pole cell formation in Smittia sp (Chironomidae, Diptera): Action spectrum and photoreversibility

The formation of pole cells (primordial germ cells) in Smittia sp can be inhibited by ultraviolet (uv) irradiation without causing significant mortality. Until 70 min after egg deposition, pole cells are suppressed by low uv doses applied to the posterior pole region. Microbeam irradiation of a target area including the oosome inhibits pole cell formation; this is not observed after irradiation of other target areas. The action spectrum for uv inhibition of pole cells shows a distinct peak at 260 nm; its shape suggests that a nucleic acid-protein complex acts as an effective target. Independent evidence for the involvement of a nucleic acid moiety is derived from the fact that uv inhibition of pole cell formation is photoreversible. The results are discussed in the context of pole cell determination by localized cytoplasmic components.

Modifying effects of ultraviolet irradiation on the development of abnormal body patterns in centrifuged insect embryos (Smittia sp., Chironomidae, Diptera)

Abstract In Smittia and other chironomid embryos, both anterior and posterior egg halves can give rise to either anterior or posterior segments. Upon various types of experimental interference, eggs may develop one of four basic body patterns: normal embryos, double cephalons, double abdomens, or inverted embryos. From a previous model of anteroposterior determination, we derive four sets of predictions for the results of combined ultraviolet irradiation and centrifugation experiments. While most of the actual results are in agreement with the predictions, some are not. Most of the discrepancies are resolved in a modified version of the model. According to the new model, anterior and posterior egg halves contain both anterior and posterior cytoplasmic determinants. These are thought to be mutually repressive, and to control an overall determination for either anterior or posterior development. Centrifugation and ultraviolet irradiation appear to affect the relative strength of anterior determinants in one or both of the egg halves, thus modifying the probabilities for the four basic body patterns to develop. Different frequencies of these patterns, which have been obtained after similar experimental treatment of different chironomid species, can be ascribed to species-specific variation in the ultraviolet sensitivity of anterior and posterior determinants.

Proteins foretelling head or abdomen development in the embryo of Smittia spec. (Chironomidae, Diptera).

Abstract The development of the segment pattern in Smittia embryos can be manipulated experimentally. Centrifugation during intravitelline cleavage leads to a mirror image duplication of most of the head in the absence of abdominal segments (“double cephalons”). Conversely, mirror image duplications of abdominal segments in the absence of head and thorax (“double abdomens”) can be generated by UV-irradiation of the anterior pole before blastoderm formation. By subsequent exposure to blue light, UV-irradiated embryos can be reprogrammed for normal development (photoreversal). We have characterized an “anterior indicator” protein (designated AI 1 ; M r ⋍ 35,000; IEP ⋍ 4.9). Its synthesis was restricted to anterior fragments of embryos during a late blastoderm stage (Bl VI ). This protein was synthesized, however, in both anterior and posterior fragments of prospective double cephalons. Conversely, this protein was synthesized neither in anterior nor in posterior fragments of UV-induced double abdomens. Upon photoreversal, the protein was synthesized again in anterior fragments. Thus, synthesis of this protein in a given fragment always indicated development of head and thorax there. Likewise, we have characterized a “posterior indicator protein” (designated PI 1 , M r ⋍ 50,000, IEP ⋍ 5.5). Its synthesis during early blastoderm stages (Bl I and Bl II ) was restricted to posterior fragments but not to pole cells in normal embryos. In UV-induced double abdomens, PI I was synthesized in both anterior and posterior fragments at stage Bl II . Photoreversal again led to restriction of PI I synthesis to posterior fragments. Thus, the synthesis of PI I in a given fragment at stage Bl II always foreshadowed the formation of an abdomen several hours before this can be discerned morphologically. The synthesis of two other proteins (designated a 1 and p 1 ) was also restricted, during certain blastoderm stages, to anterior or posterior fragments, respectively. However, UV-irradiation or centrifugation had little or no effect on the synthesis of these proteins. Conversely, programming embryos for double abdomen development by UV-irradiation caused a set of reproducible, and mostly photoreversible, changes in the pattern of proteins synthesized in anterior embryonic fragments. However, the synthesis of most of the affected proteins was not region-specific in normal embryos.

AN UNUSUAL CASE OF PHOTOREACTIVATION OBSERVED IN AN INSECT EGG (SMITTIA SPEC, CHIRONOMIDAE, DIPTERA)*

Abstract— An action spectrum for inactivation by ultraviolet (UV) radiation of Smittia eggs during intravitelline cleavage was established, taking into account wavelength‐dependent shielding of the effective targets. Under the assumption of a random distribution of the effective targets in the egg, the action spectrum displayed only one very distinct peak at 295 nm. The eggs were photoreactivable with an action spectrum similar but not identical to that found for direct photoreactivation (PR) in E. coli Indirect PR seems not involved because light was effective only after but not before UV. Temperature dependence and dose rate saturation could not be observed. The photoreactivable sector (PRS) was 0.75 after UV inactivation at 295 nm but only 0.32 after UV inactivation at 265 nm. Initial PR rates were highest after 295 nm and lowest after 265 nm. During migration of cleavage nuclei into the periplasm, when the shielding of nuclei by yolk material decreases by an order of magnitude, no corresponding increase in the sensitivity of the eggs to UV was observed. After inactivation at the blastoderm stage, when the nuclei are no longer shielded by yolk material, the PRS was also high (0.79) after UV of 295 nm but again lower (0.59) after 265 nm. These data are difficult to understand within the conceptual framework of light‐dependent enzymatic splitting of UV‐induced pyrimidine dimers in nucleic acids. Yet this type of PR seems to play a vital role in the survival of Smittia eggs under sunlight without need for pigmentation or shading.

Photoreactivation of Pyrimidine Dimers Generated by a Photosensitized Reaction in RNA of Insect Embryos (Smittia Spec.)

Insect eggs are large cells, ranging from a few tenths to several millimeters in length. The egg cell is surrounded by a vitelline membrane, and an outer shell, the chorion. The yolk-rich and opaque endoplasm contains glycoproteid spheres, lipid droplets, and glycogen particles, whereas the superficial periplasm is yolk-free. After fertilization or parthenogenetic activation, the nucleus undergoes a series of mitotic divisions within the yolk endoplasm. This period is referred to as intravitelline cleavage, although the egg cell is not cleaved. Rather, the embryo develops in a plasmodial state, containing eventually hundreds of energids, i.e. nuclei with jackets of cytoplasm. During nuclear migration stages, most energids move into the yolk-free periplasm, where they become enclosed by infoldings of the plasmalemma of the egg cell. The resulting blastoderm cells may be eventually separated from the yolk endoplasm, or may remain connected to it by cytoplasmic bridges. While cellularization is in progress, nuclear divisions may continue. Following this period of nuclear proliferation and the formation of an apparently homogeneous layer of blastoderm cells, regional differentiation begins. Many of the blastoderm cells build the originally unsegmented germ anlage while the remainder form the embryonic membranes. After gastrulation and segmentation, the embryo reaches the germ band stage which already reflects the basic organization of the larva. Recent descriptions of early Drosophila embryogenesis have been given by Fullilove and Jacobson (1978), Turner and Mahowald (1976), and Zalokar and Erk (1970). For reviews on insect development see Counce and Waddington (1972, 1973).