Oncoviral DNAs induce transposition of endogenous mobile elements in the genome of Drosophila melanogaster.

Previously, we have shown that particles of Rous sarcoma virus or cloned fragments of RSV cDNA as well as DNA of oncogenic simian adenovirus Sa7, injected into the polar plasm of early Drosophila melanogaster embryos, were able to induce, with high frequency, unstable visible mutations in different groups of genetic loci. The genetic instability of the recovered mutations, i.e., their ability to revert to normal state or to generate new mutant alleles at the affected locus, was manifest in mutant lines through several generations. The molecular analysis undertaken in this study of the yellow-scute loci region which is highly sensitive to the microinjected Sa7 DNA, and of the white locus, that frequently mutates under the influence of RSV cDNA, clearly shows that the induced mutations and reversions are accompanied by insertion/excision of endogenous mobile elements. This conclusion is confirmed by in situ hybridization experiments which demonstrate that the adenovirus DNA is able to change, though with different efficiency, the chromosomal localization of certain Drosophila retrotransposons. These results partially elucidate the molecular mechanism of the genetic instability in D. melanogaster induced by microinjection of oncoviruses into early embryos, implying that is results from mobilization of endogenous transposons which play the role of insertional elements directly causing unstable mutations.

INDUCTION OF UNSTABLE MUTATIONS IN DROSOPHILA MELANOGASTER BY THE MICROINJECTION OF ONCOGENIC VIRAL DNA INTO THE POLAR PLASM OF EMBRYOS: PROLONGED GENETIC INSTABILITY OF MUTATIONS AT THE LOBE LOCUS.

MUTATIONS IN THE LOBE LOCUS WERE PRODUCED BY MICROINJECTION OF RSV CDNA INTO EARLY EMBRYOS OF D. MELANOGASTER, AND SHOWED PROLONGED GENETIC INSTABILITY, THE EXTENT OF WHICH CHANGED SIGNIFICANTLY WITH TIME. GENETIC ANALYSIS OF LOBE-RSV MUTATIONS AND A MOLECULAR-BIOLOGICAL ANALYSIS OF STRUCTURAL REARRANGEMENTS IN THE W AND ADH LOCI, WHICH ARE FREQUENTLY MUTATED BY RETROVIRAL CDNA, SHOWED THAT THE MUTATIONS RESULTED FROM INSERTIONS ARISING IN THIS GENETIC INSTABILITY SYSTEM.

INDUCTION OF UNSTABLE MUTATIONS IN DROSOPHILA MELANOGASTER BY THE MICROINJECTION OF ONCOGENIC VIRAL DNA INTO THE POLAR PLASM OF EMBRYOS: THE INSERTIONAL NATURE OF THE MUTATIONS.

MUTATIONS INDUCED BY THE MICROINJECTION OF ADENOVIRUS SA7 DNA INTO THE POLAR PLASM OF DROSOPHILA MELANOGASTER EMBRYOS WERE SHOWN TO BE OF THE INSERTIONAL TYPE. THE INSERTED ELEMENTS RESPONSIBLE FOR THESE MUTATIONS WERE ENDOGENOUS TRANSPOSONS RATHER THAN VIRAL SEQUENCES. THUS, GENETIC INSTABILITY ACTIVATED BY ONCOVIRAL DNA RESULTED FROM THE ABILITY OF THESE DNA SEQUENCES TO INDUCE TRANSPOSITION OF MOBILE ELEMENTS IN THE RECIPIENT GENOMES.

Unstable visible mutations induced in Drosophila melanogaster by injections of oncogenic virus DNA into the polar plasm of early embryos.

When RSV DNA cloned in pBR 322 or DNA of simian adenovirus Sa7 (C8) is injected into the pole plasm of embryos of various Drosophila stocks, the progeny of 1-70% of the surviving flies display visible mutations. The mutagenesis is partially directed: the loci mutating due to retrovirus and adenovirus DNA do not overlap. The majority of resulting mutants are characterised by high instability: reversions and new mutations occur in them, which sometimes spread over the whole population ("explosive" instability). The injected sequences are revealed by dot-hybridization in the DNA of many mutant strains, but only rarely by Southern blotting procedures. The results show that the microinjection of oncovirus DNA into embryos is an approach for obtaining highly unstable strains even from wild-type stable Drosophila stocks without crosses with MR lines or the introduction of P elements. The sets of unstable mutations induced by oncovirus DNA is different from those in hybrid dysgenesis.

Avian primary erythropoiesis; accessory pathways of erythrocyte maturation in chick yolk sack.

In the population of chick primary erythroblasts the quota of the G2-phase (tetraploid) cells is greatly increased probably due to the fact that a part of erythroblasts is reversibly blocked at this phase. An increased number of G2 cells is also detected in the population of reticulocytes at later stages of embryogenesis and there is no cell-division in about half of the G2 cells until the 8th day the erythrocytes being exclusively diploid. The primary erythrocytes are probably formed from two types of precursors: normally dividing and specialized cells, and cells blocked in the G2-phase some of which do not complete mitosis before they develop into erythrocytes. Haemoglobinization is far more intensive in primary erythroid cells as compared with erythrocytes of definitive normal erythropoiesis.

Rearrangements of microinjected recombinant DNA in the genome of transgenic mice.

Previously, mouse zygotes were microinjected with the recombinant plasmid pMA3, which contains the Herpes simplex virus thymidine kinase gene attached to the promoter region of the Rous sarcoma virus (Gazaryan et al. 1984a). In the present work the pMA3 fragment with the flanking genomic sequences was isolated from the DNA of one transgenic Fo mouse by the "plasmid rescue" technique. The rescued plasmid (pMAR1) lacked all virus-specific sequences and retained only some pBR322 sequences. The flanking region at one end of the integrated pBR322-specific fragment contained a highly conserved mouse repetitive sequence. The possible mechanisms of rearrangement of foreign DNA in germ line cells are discussed.

Microinjection of simian adenovirus SA7 (C-8) DNA into the mouse zygotes: differential distribution of viral DNA in organs.

A simian adenovirus SA7 (C-8) DNA was microinjected into fertilized mouse eggs. Thirty-five mice derived from eggs injected with SA7 DNA were screened for the presence of the adenovirus genome in their liver DNA. Eighteen of these mice contained the virus-specific sequences. SA7 DNA was detected in some tissues, but in all cases, viral sequences were absent from muscle and heart DNA. Viral DNA was inherited by 50-70% of the next generation. One mouse that contained about 1 copy of SA7 DNA per haploid genome has been shown to pass it on to five generations, although the integrated viral DNA sustained a considerable structural change between F1 and F3. RNA analysis in various organs of 12 mice has shown the transcription of SA7 DNA to be very infrequent: only in the kidney of one mouse and in the spleen of another did RNA contain SA7-specific sequences.

Detection of virus-specific sequences in Drosophila melanogaster mutants induced by injection of RSV DNA into early embryos.

The injection of purified Rous sarcoma virus (RSV) (Prague strain) into Drosophila melanogaster (Oregon R line) eggs changes the fly phenotype in certain cases, and RSV-specific sequences can be identified in the Drosophila genome (ref. 1 and preceding paper). Here we have used Southern blotting to analyse in greater detail the proviral DNA present in several mutant lines of D. melanogaster produced by microinjection of intact RSV or plasmid DNA containing the viral insert. In certain populations of flies, RSV provirus was found to be incorporated into cellular DNA, and in one mutant family the unintegrated form of plasmid DNA was identified. Generally, the presence of injected genetic material in fly cells correlated with morphological changes in Drosophila.

Genetic effects of injection of Rous sarcoma virus DNA into polar plasm of early Drosophila melanogaster embryos.

Retroviral proviruses and the transposable elements of eukaryotic genomes are structurally similar. The biological significance of eukaryotic transposable elements has not been examined extensively but it is known that, like prokaryotic transposons, these elements can induce mutations in adjacent genes and cause their transposition. It is of interest to determine whether retroviral proviruses have the same mutagenic and gene transposing ability as transposable elements, particularly because the retrovirus genome is assumed to have originated from transposable elements of lower eukaryotes. The transfer of DNA sequences into animal zygotes or embryos by microinjection is a promising experimental approach for eluxidating their functions: when foreign DNAs were introduced into a mouse germ line, mutations were induced and at least in some mice, the mutation was caused by the insertion of a retroviral sequence. We have introduced Rous sarcoma virus (RSV) DNA into a germ line of Drosophila melanogaster, and describe here the resultant genetic effects.

Effect of microinjections of cAMP-dependent protein kinase subunits on macromolecular syntheses in loach Misgurnus fossilis L. embryos.

The level of cAMP and the effects of the regulatory and catalytic subunits of cAMP-dependent protein kinase of type II on protein, RNA and DNA synthesis in loach embryos were analysed. It was found that the injections of the catalytic subunit cause a sharp decrease in the rate of macromolecular synthesis while an increase in the concentration of this protein leads to the death of the embryo. Injections of the regulatory subunit result in marked stimulation of protein, RNA and DNA synthesis. The effect of the regulatory subunit on these processes is rather complex. Experiments on the transplantation of the nuclei from the embryos treated with the protein kinase subunits into normal embryos were carried out.

Dimensions of homo- and heteropolymeric sections of DNA synthesized by reverse transcription from globin mRNA matrices.

The lengths of globin messenger RNAs, isolated from rabbit and pigeon reticulocytes, and single-stranded complementary DNAs, synthesized from mRNA in presence of RNA-directed DNA-polymerase and oligo(dt) have been investigated by polyacrylamide gel electrophoresis under denaturing conditions. The cDNA preparations contained a fraction which corresponded in length to the alpha and beta chains of the mRNA used as a matrix. The lengthwise distribution of poly(A) sequences, isolated from globin mRNAs, corresponded to a statistical distribution, with a maximum at 75-80 nucleotides and with a maximum length of approximately 150 nucleotides. The lengthwise distribution of poly(dt) sequences, isolated from cDNA, corresponded to the poly(A) sequence distribution. It was concluded that: 1) cDNA is heterogeneous with respect to the length of poly(dt) sequences located at the 5' end of the molecule; 2) there was no selective use as primer of only that oligo(dt) which was located within the complex formed with the 5' end of the mRNA poly(A) sequence; 3) the heterogeneity of poly(DT) corresponded qualitatively to two models of template-primer interaction: selective use as a true primer of the oligonucleotide located in the complex formed with the 3' end of mRNA, and a statistical distribution of primer along the poly(A) sequence; and 4) the heterogeneity of the homopolymeric section of the cDNA may affect the kinetics of hybridization with mRNA, which possibly explains the previously observed [1] anomalous dependence of the hybridization rate on time.