RNA synthesis during early embryogenesis of mass cultured highly synchronized coleopteran embryos

Summary Bruchidius embryos are shown to be well suited for biochemical studies during early embryogenesis. Mass cultivation is easy, and highly synchronized embryos can be obtained in large numbers (10⁴–10⁵ eggs). A method for in vivo incubation is described which allows the labelling of newly synthesized RNA. The kinetics of³H-ruidine uptake, phosphorylation and incorporation into RNA are presented. By autoradiography, the distribution of newly synthesized RNA is shown. Thereby, stage-specific differences were found in the labelling pattern of vitellophage nuclei, of blastoderm nuclei and of the nuclei of pole cells. The labelling of the cytoplasm remains weak until cellular blastoderm is formed. During late blastoderm and at gastrulation this label increases markedly. Gel electrophoresis of isolated RNA shows that at cellular blastoderm formation most of the label occurs in a region between 18 S and 7 S. Later on, at the onset of gastrulation, the³H-uridine incorporation found in isolated RNA is raised about 10 fold and rRNA synthesis becomes prominent. In a chase experiment, the processing of precursor RNA molecules into shorter RNA species, especially into mature rRNA and 5S RNA, is shown. The advantages of theBruchidius embryo for the biochemical analysis of early RNA synthesis and the regulation of rRNA synthesis in insect embryos are discussed.Dedicated to Professor Dr. Dr. h. c. Bernhard Rensch at the occasion of his 80th birthday     

Rapid down regulation of insulin receptors in adipocytes artifact of the incubation buffer

Rat adipocytes were incubated with 15 nM insulin in different buffers at 37°C. The cells were washed and reincubated at 16°C in the presence of 18 pM A14-[¹²⁵I]monoiodoinsulin to determine the insulin receptor concentration. After incubation for 2 h in Tris buffer the binding decreased to about 30 %, whereas no decrease was found after incubation in Hepes, phosphate or bicarbonate buffers. Binding of tracer insulin reached a constant level by 45 min in Hepes buffer at 37°C, whereas it continued to increase in Tris buffer. Washout of tracer insulin after incubation in Tris buffer at 37°C showed a large, slowly dissociable fraction. It is suggested that the rapid down regulation of insulin receptors $\text{in}\text{vitro}$ is an artifact of the Tris buffer and that the phenomenon is due to a slowly reversible occupancy of a receptor pool with unlabelled insulin.     

Stimulation of DNA synthesis and myo-inositol incorporation in mammalian cells

Phosphatidylinositol (PI) synthesis and its role in controlling the cell cycle has been investigated using fibroblasts and liver cells in culture. PI synthesis as measured by incorporation of [³H]-myo-inositol into trichloroacetic acid precipitable material during 0–60 min after serum or growth factor stimulation of serum-starved cells is increased in primary fetal rat liver cells, rat embryo fibroblasts, and 3T3 mouse cells. In contrast, growth stimulation of 3T3 cells and hepatocytes rendered quiescent in G₁ by amino acid starvation is not accompanied by increased incorporation of [³H]-myo-inositol into trichloroacetic acid precipitable material. This suggests that those cells might be arrested at a different point in G₁ than cells arrested by serum depletion. Inhibition of PI synthesis by δ-hexachlorocyclohexane (HCH), a steric analog of myo-inositol, during early times (e.g., 0–4 hr) after growth stimulation, reversibly blocks initiation of DNA synthesis in 3T3 cells. The results support the idea that increased PI synthesis in response to growth stimulation in the cell types studied here is a prerequisite for progression through G₁ and subsequent entry into S phase.     

Excitation parameters of the repetitive firing mechanism from a statistical evaluation of nerve impulse trains

Repetitive firing of single tonic neurones is modeled to include in detail both membrane excitation kinetics and electrotonic effects due to membrane non-uniformities in the impulse encoder region. The model is evaluated dynamically and compared with similar data obtained from the crayfish stretch receptor neuron. Two dynamic techniques utilizing small amplitude sinusoidal signals are employed. One technique is used to fix the values of two parameters which relate to the electrotonic control of membrane potential in the interspike interval and to the relaxation time of the K-conductance during repetitive firing. The other technique is employed as a consistency check. The dynamics are particularly sensitive to the K-channel relaxation time in the interspike interval.Research supported by NSF grant BNS 77-22532 and Public Health Service Grant EY 00293. Computer facilities were made available by a grant from the Air Force Office of Scientific Research (AFOSR-1221) and by the University of Minnesota Computer Center     

Differences in the number of nucleolus organizers in Chironomus tepperi shown by in situ hybridization

Two stocks of Chironomus tepperi could be isolated. One stock, N(IV)⁺, contains nucleolus organizers in chromosome I and IV, whereas the other one, N(IV)^–, shows only one nucleolus in chromosome I. It is demonstrated by in situ hybridization with radioactive rRNA that the absence of the nucleolus in chromosome IV of stock N(IV)^– is not related to an inactivation of the nucleolar DNA, as might have been suggested, but is due to the lack of ribosomal cistrons.     

The isolation of IS1 and IS2 DNA

Summary DNA of the IS-elements IS1 and IS2 was prepared by digestion of appropriate heteroduplex molecules with endonuclease S1, followed by sucrose gradient centrifugation or gel electrophoresis. The material obtained is homogeneous with regard to size. The length of IS1 DNA is 820±65 nucleotides, the length of IS2 DNA is 1.350±70 nucleotides. IS1 DNA is not cleaved by the restriction endonucleases Eco R1, Hind II or Hind III. IS2 DNA is cleaved once by each of the two latter enzymes. The buoyant density determined by equilibrium centrifugation of Hg-complexes in Cs₂SO₄ corresponds to a GC content of approximately 50%. Labelling with polynucleotide kinase indicates that both IS DNA's have a guanosyl residue at both of their 5-termini.     

Effects of temperature on the rates of oxygen consumption during morphogenesis and diapause in the egg stage of Letopterna dolobrata (Heteroptera,Miridae)

Summary Oxygen consumption at 25°C was measured continously throughout the egg stage of Leptopterna dolobrata (more than 9 months).The rate of O₂-uptake (l O₂/100 eggs · 1 h) is low in freshly laid eggs. Maintaining the eggs at a constant temperature of 16°C, respiration rises abruptly from the first day after oviposition and continues rising steadily for 3 days, reaching an average value of 1.4 l. Oxygen consumption persists at or near this high level during the developmental phase of prediapause, which lasts about 15 days. After some days of oscillating high and low values, respiration decreases, and from the 24th day a low level (0.3–0.4 l) is reached. If the eggs are incubated at 16°C continuously, this low diapause-level is maintained until the end of the experiments (42 weeks) and diapause is terminated in a few eggs only.A significant increase in the success of hatching is obtained by exposing the eggs to a sufficient period of chilling.24 groups of diapausing eggs were chilled at 5°C for certain periods (10, 18, 22, 26, 31, and 34 weeks) and afterwards transferred to 16°C and re-incubated. The changes of their O₂-uptake at 25°C were traced throughout their chilling and successive re-incubating periods.Oxygen consumption is greatly accelerated during the cold treatment of the eggs. The low values of the diapause-level are raised progressively during the first 6 weeks of chilling. After this primary rapid increase, respiration remains at a level 5-times as high as the diapause values over a period up to the 25th week at 5°C. This is almost exactly the duration of mesodiapause (6 months).The rates of O₂-uptake during the subsequent re-incubation at 16°C depend on the extent of chilling. The ability of diapause-breaking is correlated to the rates of O₂-uptake, measured after setting of re-incubation. If respiration never decreases by the onset of re-incubation, diapause is terminated in most of the eggs, and the rates of O₂-uptake increase as re-incubation goes on towards the emergence of the larvae (postdiapause period).A preliminary interpretation of the cold-stimulated O₂-uptake in diapausing Leptopterna-eggs is given.Dedicated to Prof. Dr. H. Precht (Kiel) on the occasion of his 65th birthday     

Structural studies of the polysaccharide antigen of Eubacterium saburreum, strain 49.

The polysaccharide antigen produced by Eubacterium saburreum, strain L 49, is composed of D-glycero-D-galacto-heptose and a new sugar, tentatively identified as 6-deoxy-D-altro-heptose. It contains chains of alternating (1 leads to 3)- and (1 leads to 6)- linked beta-D-glycero-D-galacto-heptopyranosyl residues, the latter being substituted with 6-deoxy-alpha-heptofuranosyl groups at O-3. The polysaccharide further contains 0-acetyl groups, linked to O-7 of part of the heptosyl residues and to O-2 of part of the 6-deoxyheptosyl groups.