Herbert Jäckle

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.

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.

Spatial distribution of abundant proteins in oocytes and fertilized eggs of the Mexican axolotl (Ambystoma mexicanum).

Two-dimensional protein patterns were compared from sections along the longitudinal axis of oocytes and fertilized eggs of the Mexican axolotl (Ambystoma mexicanum). Only a few differences were observed between four different sections through both oocyte and fertilized eggs. A set of proteins (14 out of 120 proteins) were found that reside only in the germinal vesicles (GV) of the fully grown oocyte. Two of these were observed exclusively in the vegetal half, and one in the animal half after GV breakdown, while other proteins were randomly distributed within the fertilized egg. One cytoplasmic protein was present only in the vegetal half of the mature oocyte and became present also in the animal half of the fertilized egg. Additional proteins were observed in all transverse sections of both mature oocyte and fertilized eggs. It is proposed that these proteins are modified rather than newly synthesized proteins.

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.

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.

Actin messenger in maternal RNP particles from an insect embryo (Smittia spec., Chironomidae, Diptera)

SummaryActin synthesis was observed in early embryos of the chironomid midgeSmittia spec. The criteria used to identify newly synthesized actin include comigration with purified actin in two-dimensional gel electrophoresis, and peptide mapping after limited proteolysis. Actin was also identified after in vitro translation of RNA or salt washed RNP-particles isolated from newly deposited eggs. While intact RNP-particles stimulated protein synthesis only poorly, the rate of synthesis increased considerably after partial or complete removal of proteins from RNP-particles. Actin was synthesized both during advanced stages of oogenesis and during early embryogenesis. In embryos during intravitelline cleavage actin synthesis was insensitive to inhibitors of transcription (actinomycin D, α-amanitin). Actin was found everywhere along the longitudinal axis of the embryo using a procedure which allowed simultaneous sectioning of a large number of embryos.

Two-dimensional gel analysis after removal of major proteins reveals stage-dependent proteins in early insect development

Protein synthesis occurring during development, or in the various tissues of eucaryotic organisms, is commonly analysed by high resolution twodimensional(2D).gel electrophoresis [l] or by immunological techniques [2]. Both techniques, however, are limited in detecting small amounts of proteins in the presence of abundant other proteins [2,3]. Consequently, few tissue-specific and/or stage-dependent proteins have been reported [3-81. For insect embryogenesis, neither qualitative nor even major quantitative differences have been observed in the patterns of proteins synthesized before the onset of embryonic mRNA synthesis around blastoderm formation [2,9,20]. This paper describes removal of major proteins by antibody precipitation followed by 2D-gel analysis of the non-precipitated proteins. Using this method, we observed differential protein synthesis at two early stages of insect development, not detected by either immunological techniques or 2D-gel analysis [2,9,10]. Moreover, our results suggest translational control for maternal mRNA already present in the insect egg.

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).

PHOTOSENSITIZED FORMATION OF RNA-PROTEIN CROSSLINKS IN AN INSECT EGG (SMITTZA SPEC., CHIRONOMIDAE, DIPTERA)*

Abstract—Upon irradiation of Smittia eggs with ultraviolet light (UV), the extractability of RNA with phenol decreased. The strongest decrease was observed after irradiation at 295 nm wavelength. After trypsin treatment, RNA could be recovered to the same degree as from unirradiated eggs. The extractability of RNA from irradiated eggs was not enhanced by formamide. dimethyl sulfoxide. high salt concentration or heat treatment. The results suggest that the UV‐mediated formation of RNA‐protein crosslinks in Smittia eggs involves the action of a photosensitizer.