Induction of unstable mutations in Drosophila melanogaster by microinjection of DNA from oncogenic viruses into the embryo polar plasma. I. Effect of the embryo genotype

We have studied the spectrum and nature of mutations induced by oncogenic virus DNA injections into wsn, T-007 line of embryos, and those of the first generation hybrids obtained after crossing the T-007 line males with the Oregon R wild line females (hybrid disgenesis). Each line is shown to have a special group of "hot" sites mutating with high frequency under the effect of the oncovirus DNA injected.     

Biological effect of human erythropoietin in transgenic mice]

Transgenic mice were obtained inheriting the human erythropoietin gene under the control of viral regulatory elements. The reliable difference in haematocrit, the content of haemoglobin and percentage of reticulocytes in peripheral blood were not revealed. The level of serum erythropoietin in transgenic mice is several fold higher than in control mice. The increased pool of erythroid cells was observed in the bone marrow of transgenic mice, especially of normoblasts (3-fold) and reticulocytes (4,5-fold).     

DNA synthesis and content and the accumulation of total protein and hemoglobin during the differentiation of primary erythroid cells in chickens

Using cytophotometric and autoradiographic methods, it was shown that on days 2-3 of embryogenesis primary erythroid cells (PEC) divided actively. The distribution of erythroblasts (EB) according to their DNA content is not, however, typical of a proliferating population: it contains an unusually large number of 4c cells resulting from the cell cycle arrest at the G2 phase. It is established that reticulocytes (RC) do not divide and are arrested at G1 or G2 phases, since they do not incorporate 3H-thymidine after their formation is complete and their DNA contents are strictly confined to either 2c or 4c. All types of PEC include a large number of cells containing H2c DNA which is due either to the cell cycle arrest at the S phase, or to the formation of accessory nuclei. All PECs have much higher contents of hemoglobin and total protein than do adult hen erythrocytes (EC). Hemoglobin and total protein contents of H2c and accessory nuclei containing cells are much higher than those in 2c-cells. We have calculated that adult birds and embryos contain the same amount of hemoglobin per gram of weight, but the quantity of red blood cells in the former is ten times higher. A conclusion is drawn that proliferation and cytodifferentiation regulation mechanisms are directed, in primary erythropoiesis, to intense hemoglobinization of the cells, and, in adult erythropoiesis, to increasing their number. In both the cases homeostatic regulation of erythropoiesis works.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characteristics of the pathways of erythroid cell differentiation in birds in anemia

A comparative study has been made of erythroid cell development pathways in the peripheral blood of pigeons during severe, moderate and weak forms of anaemia. Three modes of erythrocyte formation from bone marrow precursor are described: 1. A reserve erythropoiesis--the principal process during severe anaemia; the bone marrow precursors are basophylic erythroblasts which are reversibly blocked in phase G2 of the cell cycle; in results the rapid, increase of erythrocyte population above the normal level, although the cells have 25-30 per cent deficiency in haemoglobin content. 2) A mode of erythropoiesis, whose precursors are proliferating polychromatophylic erythroblasts; this is the principal mode of erythropoiesis at the moderate anaemia, leading to restoration of the normal quantity of erythrocytes with a normal haemoglobin content. 3) A mode of erythropoiesis with proliferating orthochromatic erythroblasts being precursors (which do not divide normally); this is the principal mode during the weak anaemia to result in a slow restoration of the number of erythrocytes with an excess in haemoglobin content. It is shown that regulation of the restoration processes during anaemia are characterized by a specific combination of cell proliferation and differentiation.     

Reassociation of interspersed moderate DNA repeats

Reassociation kinetics of the fragments of DNA consisting of interspersed repetitive and non-repetitive nucleotide sequences is considered in this paper. Based on the model, suggested by Gavrilov and Mazo (Mol. biol., 11, 101 1977), which takes into account the random DNA shearing, both reassociation kinetics of the total DNA in the region corresponding to interspersed repeat reassociation and that of the isolated preparation of interspersed repetitive sequences are calculated. In both cases influence of the repeat length on the reassociation rate is demonstrated. The estimation of the repetition frequency of rare repeats from pigeon genome is specified using calculations performed.     

Induction of unstable mutations in Drosophila melanogaster by microinjections of oncogenic virus DNA into the embryo polar plasma. Prolonged genetic instability of mutations at the lobe locus]

Mutations Lobe induced by the microinjections of RSV cDNA into Drosophila melanogaster early embryos are characterized by permanent genetic instability; the level of this instability is being changed in time. Based on the results of genetic analyses of Lobe mutations and molecular analysis of white and ADH mutations induced at high frequency in this system of gene instability, we supposed that unstable mutations which arose under the influence of retroviral cDNA are of the insertional nature.     

Globin-coding sequences in the nuclear 28S pre-mRNA and in the cytoplasmic RNA of pigeon erythroid cells

The steady-state content of globin-coding sequences in nuclear and cytoplasmic RNA of pigeon erythroid cells was estimated by hybridization in the excess of nuclear 28S RNA and cytoplasmic poly(A) + RNA with [3H]DNA, synthesized on globin mRNA. Sequences of 9S globin mRNA are found in 0.06% of molecules of non-ribosomal 28S nuclear RNA (pre-mRNA) of erythroblasts and in 0.5% of molecules of non-ribosomal 28S nuclear RNA of reticulocytes. The content of globin mRNA in erythroblast cytoplasm is, respectively lower than in that of reticulocytes.     

Reactivation of the nuclei of reticulocytes and erythrocytes in heterokaryons]

The reactivation of the nuclei of erythrocytes, reticulocytes and bone marrow cells has been studied by means of hybridization of the pigeon erythroid cells with the human embryonic cells A1. The process of reactivation of the erythroid nucleus was shown to depend on the stage of erythroid cell differentiation. The nuclei of cells at the earlier stages of differentiation give a higher percentage of heterocaryons (40%, 70%) than those of more mature cells (9%). The activated nuclei of immatur cells formed nucleoli already 24 hrs, whereas those of mature erythrocytes only 3-5 days after the cell fusion.     

Interaction of dipeptydil peptidase IV with amyloid peptides

The aggregates of amyloid beta peptides (Aβs) are regarded as one of the main pathological hallmarks of Alzheimer’s disease (AD). An imbalance between the rates of synthesis and clearance of Aβs is considered to be a possible cause for the onset of AD. Dipeptidyl peptidases II and IV (DPPII and DPPIV) are serine proteases removing N-terminal dipeptides from polypeptides and proteins with proline or alanine on the penultimate position. Alanine is an N-terminal penultimate residue in Аβs, and we presumed that DPPII and DPPIV could cleave them. The results of present in vitro research demonstrate for the first time the ability of DPPIV to truncate the commercial Aβ40 and Aβ42 peptides, to hinder the fibril formation by them and to participate in the disaggregation of preformed fibrils of these peptides. The increase of absorbance at 334 nm due to complex formation between primary amines with o-phtalaldehyde was used to show cleaving of Aβ40 and Aβ42. The time-dependent increase of the quantity of primary amines during incubation of peptides in the presence of DPPIV suggested their truncation by DPPIV, but not by DPPII. The parameters of the enzymatic breakdown by DPPIV were determined for Aβ40 (K_m = 37.5 μM, k_cat/K_m = 1.7 × 10³ M⁻¹sec⁻¹) and Aβ42 (K_m = 138.4 μM, k_cat/K_m = 1.90 × 10² M⁻¹sec⁻¹). The aggregation-disaggregation of peptides was controlled by visualization on transmission electron microscope and by Thioflavin-T fluorescence on spectrofluorimeter and fluorescent microscope. DPPIV hindered the peptide aggregation/fibrillation during 3-4 days incubation in 20 mM phosphate buffer, pH 7.4, 37 °C by 50–80%. Ovalbumin, BSA and DPPII did not show this effect. In the presence of DPPIV, the preformed fibrils were disaggregated by 30–40%. Conclusion: for the first time it was shown that the Aβ40 and Aβ42 are substrates of DPPIV. DPPIV prohibits the fibrillation of peptides and promotes disaggregation of their preformed aggregates.