Artificial and epigenetic regulation of the I factor, a nonviral retrotransposon of Drosophila melanogaster.

The I factor (IF) is a LINE-like transposable element from Drosophila melanogaster. IF is silenced in most strains, but under special circumstances its transposition can be induced and correlates with the appearance of a syndrome of female sterility called hybrid dysgenesis. To elucidate the relationship between IF expression and female sterility, different transgenic antisense and/or sense RNAs homologous to the IF ORF1 have been expressed. Increasing the transgene copy number decreases both the expression of an IF-lacZ fusion and the intensity of the female sterile phenotype, demonstrating that IF expression is correlated with sterility. Some transgenes, however, exert their repressive abilities not only through a copy number-dependent zygotic effect, but also through additional maternal and paternal effects that may be induced at the DNA and/or RNA level. Properties of the maternal effect have been detailed: (1) it represses hybrid dysgenesis more efficiently than does the paternal effect; (2) its efficacy increases with both the transgene copy number and the aging of sterile females; (3) it accumulates slowly over generations after the transgene has been established; and (4) it is maintained for at least two generations after transgene removal. Conversely, the paternal effect increases only with female aging. The last two properties of the maternal effect and the genuine existence of a paternal effect argue for the occurrence, in the IF regulation pathway, of a cellular memory transmitted through mitosis, as well as through male and female meiosis, and akin to epigenetic phenomena.

Rapid evaluation in Escherichia coli of antisense RNAs and ribozymes.

The characterization of a simple bacterial system using commercially available plasmids and strains, developed to assess the effectiveness of trans-acting (antisense RNA and ribozymes) RNA in Escherichia coli, is reported. This system was used to test various trans-acting RNA molecules against the expression of the I factor, a functional transposable element from Drosophila melanogaster. For this target, results indicate that antisense RNA efficiency depends on the hybridization length between sense and antisense RNA. The introduction of a single hammerhead ribozyme within the antisense RNAs does not increase its inhibitory activity. These predictions were subsequently confirmed in Drosophila melanogaster.

The full-length transcript of the I factor, a LINE element of Drosophila melanogaster, is a potential bicistronic RNA messenger.

The I factor of Drosophila melanogaster is a retrotransposon of the LINE superfamily. The I factor displays two non-overlapping open reading frames (ORFs) that have the potential to encode for a nucleic acid-binding protein (ORF1) and a reverse transcriptase (ORF2). Retrotransposition of the I factor has been demonstrated and a putative full-length RNA intermediate has been identified. No other transcript from functional I factor has ever been described, suggesting that the full-length RNA is also used as a messenger. Here we report that a bicistronic RNA which conserves the ORF1-ORF2 organization of the I factor transcript is a template for ORF2 translation in vivo. We further demonstrate that the first AUG of ORF2 initiates translation, but efficiency of this initiation increases approximately 200 fold when ORF1 is deleted. Our results show that the I factor transcript may be used to translate both ORFs from their own initiation codons at different rates. Various mechanisms of translation are proposed.

Germ-line expression of a functional LINE from Drosophila melanogaster: fine characterization allows for potential investigations of trans-regulators.

The I factor (IF) is a functional non-viral retrotransposon, or LINE, from Drosophila melanogaster. It is mobilized in the germ-line of dysgenic SF females during I-R hybrid dysgenesis. In previous papers (Lachaume et al., Development 115: 729-735, 1992; Lachaume and Pinon, Mol. Gen. Gen. 240: 277-285, 1993) we used a transgenic fusion between the 5' part of the IF and the lacZ gene to characterize IF expression and its regulation. This I-lacZ transgenic fusion expresses beta-galactosidase activity during oogenesis. We established a Drosophila line bearing four transgenic insertions (the 4I-lacZ line) and got new insights about IF expression: (1) I-lacZ expression is proportional to the copy number of transgenes present in the genome, (2) the expression occurs just before or when meiosis begins, (3) this expression seems to be subjected to a variegation effect within the germ-line cells, (4) the transgenic activity is mainly directed toward the decondensed chromatin of nurse cells. The close relationship between I factor expression and oogenesis led us to investigate the role played by genes expressed during oogenesis on I factor expression. We present recent data indicating that mutants which interfere with oogenesis can also affect I factor expression. From this data we propose an original screen using the 41-lacZline to detect identified mutations which also affect I factor expression.

Germ-line expression of the I factor, a functional LINE from the fruit fly Drosophila melanogaster, is positively regulated by reactivity, a peculiar cellular state.

I factor is a functional LINE (long interspersed nucleotidic element) which is mobilized in the germ-line of dysgenic SF females during I-R hybrid dysgenesis. Such females are obtained when an oocyte from a reactive stock, devoid of I factors but characterized by a level of reactivity, i.e. its potential for hybrid dysgenesis, is fertilized by a spermatozoon from an I factor-containing inducer stock. In a previous paper we described the expression of an I factor-lacZ fusion. Expression was detected in the ovaries of reactive and dysgenic flies only. In this paper we show that this transgenic activity can be quantified and depends upon the maternally inherited reactivity. Reactivity is not just a permissive state and modifiers of the reactivity level such as heat treatment and ageing change the level of expression of our transgenic fusion accordingly. Moreover, ageing through generations has the same cumulative and reversible effect on both reactivity and I factor expression. Using our fusion as a test for reactivity we show that the silencing of I factor after its introduction into a reactive genome may not be established in a single generation.

Spatial and temporal expression of the I factor during oogenesis in Drosophila melanogaster.

The I factor is a functional non-viral retrotransposon, or LINE, from Drosophila melanogaster. Its mobility is associated with the I-R hybrid dysgenesis. In order to study the expression pattern of this LINE in vivo, a translational fusion between the first ORF of the I factor and the lacZ gene of Escherichia coli has been carried out and introduced in the genome of reactive (R) flies. Homozygous transgenic Drosophila lines have been established and analysed. ORF1 expression is limited to germ-line cells (nurse cells and oocyte) between stage 2 and 10 of oogenesis. No somatic expression is found. Position effects may limit the level of expression of a given transgene but do not modify its basic pattern of expression during the development of the fly. This reproducible control demonstrates both that I factor is driven by its own promoter, probably the internal one suggested by Mizrokhi et al. (Mizrokhi, L.J., Georgevia, S.G. and Ilying, Y.V. (1988). Cell 54, 685-691), and that tissue-specific regulatory sequences are present in the 5' untranslated part of the I factor. The nuclear localization of the fusion protein reveals the presence of nuclear localization signals (NLS) in the ORF1-encoded protein correlating with the possible structural and/or regulatory role of this protein. This expression is restricted to dysgenic and reactive females, and is similar in the two conditions. All the results obtained in this work suggest that I factor transposition occurs as a meiotic event, between stage 2 and 10 of the oogenesis and is regulated at the transcriptional level. It also appears that our transgene is an efficient marker to follow I factor expression.

Host range specificity of polyomavirus EC mutants in mouse embryonal carcinoma and embryonal stem cells and preimplantation embryos.

New polyomavirus mutants (PyEC-C) selected on LT1 cells and exhibiting a strong cytopathic effect in all embryonal carcinoma (EC) cell lines tested have been isolated. They were derived by a sequence duplication event from a new multiadapted mutant isolated in PCC4 cells. A quantitative analysis of viral DNA replication and transcription in 3T6 and EC cell lines was performed to compare PyEC-C mutants and PyEC mutants previously isolated on F9 or PCC4 cell lines. Analysis of the results indicated that PyEC-C mutants were more efficient in all EC cell lines tested than all other PyEC mutants; on the contrary, they were less adapted to 3T6 cells than wild-type polyomavirus. In both 3T6 and EC cells, uncoupling between early transcription and viral DNA replication was observed; different viruses were shown to replicate with the same efficiency, while their levels of early transcripts differed by two orders of magnitude. Attempts to correlate the genome structure of the mutants with their biological properties indicate that duplication of protein-binding sequences is not the only event responsible for their phenotype. PyEC mutants were also analyzed with respect to their interactions with early mouse embryos and embryonal stem (ES) cell lines derived from the inner cell mass of blastocysts. They showed different degrees of expression in ES cells and preimplantation embryos. ES cells were most efficiently infected and lysed by mutants which exhibit both a multiadapted and a lytic phenotype in EC cells. Preimplantation embryos were not permissive to any PyEC mutants. However, EC-multiadapted mutants were infectious in blastocysts after two days of in vitro culture.

[Specific immunotherapy using Alpha-Fraction-Retard-D. pteronyssinus. Double-blind study in asthma]. / Immunothérapie spécifique par l'alpha-fraction-retard-D. ptéronyssinus. Etude en double aveugle dans l'asthme.

A multicenter, double blind clinical study on two homogeneous, treated and untreated groups, has allowed an objective appreciation of the results of specific immunotherapy using the alpha-fraction of D. pteronyssinus in asthma. A statistically significant difference was seen of the consumption of drugs in the treated group and in infants of less than 5 years (p = 0.047). Tolerance, evaluated as a function of local and secondary effects was excellent and comparable in the two groups.

Common features of polyomavirus mutants selected on PCC4 embryonal carcinoma cells.

The genomic rearrangements of six polyomavirus mutants selected on PCC4 embryonal carcinoma cells have been compared and their common characteristics pointed out. All mutants show a duplication which includes at least the adenovirus type 5 (Ad5) E1A-like enhancer core sequence plus a deletion of variable size and location. The presence of the second enhancer core sequence, the SV40-like enhancer, is not required for expression of the PyEC PCC4 phenotype. Two of these mutants are also able to express polyomavirus T antigen on F9 and LT1 cells. Multiadaptation seems to require the duplication of the Ad5 E1A-like core sequence, the maintenance of the SV40-like core sequence and a local change in DNA stability.

Isolation of polyomavirus mutants multiadapted to murine embryonal carcinoma cells.

Polyomavirus mutants were isolated from PCC4 embryonal carcinoma cells infected with a variant strain of polyomavirus (ev 1001h) and were found to contain a tandem duplication overlapping the enhancers and the origin of replication. These mutants were able to infect several lines of embryonal carcinoma cells, including PCC4, F9, and LT1. The sequence and structure of one of these mutants are presented and compared with those of other PyEC PCC4 mutants previously described.