Two distinct domains of Bicoid mediate its transcriptional downregulation by the Torso pathway.

The transcriptional activity of the Bicoid morphogen is directly downregulated by the Torso signal transduction cascade at the anterior pole of the Drosophila embryo. This regulation does not involve the homeodomain or direct phosphorylation of Bicoid. We analyse the transcriptional regulation of Bicoid in response to the Torso pathway, using Bicoid variants and fusion proteins between the Bicoid domains and the Gal4 DNA-binding domain. We show that Bicoid possesses three autonomous activation domains. Two of these domains, the serine/threonine-rich and the acidic domains, are downregulated by Torso, whereas the third activation domain, which is rich in glutamine, is not. The alanine-rich domain, previously described as an activation domain in vitro, has a repressive activity that is independent of Torso. Thus, Bicoid downregulation by Torso results from a competition between the glutamine-rich domain that is insensitive to Torso and the serine/threonine-rich and acidic activation domains downregulated by Torso. The alanine-rich domain contributes to this process indirectly by reducing the global activity of the protein and in particular the activity of the glutamine-rich domain that might otherwise prevent downregulation by Torso.

I-R system of hybrid dysgenesis in Drosophila melanogaster: analysis of the mitochondrial DNA in reactive strains exhibiting different potentials for I factor transposition.

In the I-R hybrid dysgenesis system, Drosophila melanogaster strains fall into two categories denoted inducer (I) and reactive (R). Among the reactive strains we can distinguish strains with weak, medium or strong reactivity levels. These levels are inherited in a complex way involving both chromosomal and nonchromosomal determinants, the nonchromosomal determinant being mainly maternally inherited. We were interested in determining the molecular basis of this maternal transmission. In this article we analyse the possible implication of the mitochondrial DNA in the determination of the reactivity levels. The mtDNA was analysed in lines with very different reactivity levels with the aim of correlating sequence differences with reactivity levels. The mtDNA was analysed by sequencing and restriction fragment length. No correlation was established between reactivity level and mtDNA sequence. This may favour the hypothesis that epigenetic changes would be responsible for the different reactivity levels and their transgenerational transmission.

Sequence of the novel joints present in the amplified DNA of N-phosphonacetyl-L-aspartate resistant Drosophila cells: implication on the mechanisms of amplification in these cells.

We have previously shown the presence, in the amplified DNA of a Drosophila cell line resistant to N-phosphonacetyl-L-aspartate (PALA), of two units of 150 kb and 120 kb respectively duplicated and amplified. The two joints (J1 and J2) linking these units as well as their respective wild-type counterparts have been sequenced. Sequence analysis indicates that a region of the Drosophila genome which corresponds to the proximal boundary of the 150 kb unit is common to both joints. In addition to this common region, the J1 junction possesses a 26-nucleotide sequence belonging to the J2 junction. This indicates that the J2 junction was the first formed, and that J1, therefore, results from recombination between J2 and a region of the wild-type genome 120 kb distal to J2. Sequence analysis also reveals that the joints result from illegitimate recombination between unrelated regions. AT-rich sequences, strand bias composition and putative topoisomerase I and II sites were found in at least one of the two parental sequences involved in the formation of the joints. On the basis of these results we can hypothesize that after two illegitimate recombinations between sister chromatids, leading first to J2 and then to J1, the amplification may have arisen by a series of homologous (unequal crossing-over) or illegitimate recombinations, or by an intrachromosomal rolling circle.

Structural organization and expression of amplified chromosomal sequences, which include the rudimentary gene, in cultured Drosophila cells resistant to N-(phosphonacetyl)-L-aspartate.

We have used 160 kilobases of cloned Drosophila genomic DNA from the rudimentary (r) region to examine the organization of amplified DNA in Drosophila cells resistant to 10 mM N-(phosphonacetyl)-L-aspartate (PALAr cells) obtained by stepwise selection. Evidence for the direct tandem linkage of the amplified sequences is presented. The pattern and intensity of amplified bands as well as the presence of novel junctions in the DNA sequence of PALAr cells indicate that there are two types of units of 150 and 120 kilobases long. The sequence of the smaller unit is entirely included within the larger one. The longer of the two units is present twice while the shorter one is amplified eightfold as compared to the level of the relevant DNA sequences in the wild-type. These data are consistent with a model in which successive crossing-over events over several cell cycles lead to amplification of the selected r gene and flanking sequences. However, these data can also be accounted for by a totally different mechanism in which multiple copies of DNA are generated by rolling circle replication. Transcription units other than the r gene are present within the 150 kilobase region of amplified DNA. These are found to be overexpressed in PALAr cells, though some transcripts are underrepresented relative to the copy number of the corresponding coding sequences.

4-Quinolone antibiotics: positive genotoxic screening tests despite an apparent lack of mutation induction.

The effects of different 4-quinolone antibiotic derivatives (4-Qs) in a number of short-term tests commonly employed for the evaluation of genetic toxicity were studied. Incorporation of [3H]thymidine into mitogen-stimulated peripheral blood lymphocytes was strongly enhanced at a low concentration (1.56 micrograms/ml) for most of the tested 4-Qs, whereas DNA strand breakage in lymphoblastoid cells was evident only for ciprofloxacin (10 micrograms/ml and upwards), ofloxacin (80 micrograms/ml) and norfloxacin (160 micrograms/ml). Ciprofloxacin induced a significant amount of unscheduled DNA synthesis, but was found to be negative in a shuttle vector plasmid mutation test. Ciprofloxacin (80 micrograms/ml) did not inhibit enzymes involved in the early steps of pyrimidine biosynthesis. Cell growth was slightly depressed at a concentration of 20 micrograms/ml, becoming marked at 80 micrograms/ml. In conclusion, this study seeks to contribute to an improved evaluation of genotoxic screening test data, by focusing attention on the conflicting effects imposed by the 4-Qs on a battery of such tests.

Overproduction of the first three enzymes of pyrimidine nucleotide biosynthesis in Drosophila cells resistant to N-phosphonacetyl-L-aspartate.

Drosophila cells were treated in vitro with N-phosphonacetyl-L-aspartate (PALA) which is a specific inhibitor of aspartate transcarbamylase, the second enzyme of the pyrimidine biosynthetic pathway. By stepwise selection using increasing amounts of this inhibitor, PALA-resistant (PALAr) stable clones have been isolated. Enzymatic activities of aspartate transcarbamylase, carbamyl phosphate synthetase and dihydro-orotase, borne by the same multifunctional protein, CAD, are increased 6-12-fold in these resistant clones compared with parental cells. The aspartate transcarbamylase in PALAr cells is shown by physical, kinetic and immunological criteria to be normal. The data from immunotitration and immunoblotting experiments indicate that the increased enzyme activities result from the overproduction of CAD.

Hormonal control of transcription in higher plants.

1. Nucleolar RNA polymerase Ib obtained from auxin-treated lentil roots exhibits a higher transcriptional activity than the enzyme obtained from control roots. This difference is due to a change in the enzyme properties after auxin treatment. It is suggested that the hormonal effect is mediated by a factor that changes the molecular properties of nucleolar RNA polymerase. 2. Four fractions, alpha, beta, gamma and delta, that stimulate the activity of RNA polymerase Ib, have been extracted from lentil roots. Two of them, gamma and delta have been studied. Factor delta can stimulate nucleolar polymerase Ib and the nucleoplasmic enzyme II equally well, while factor gamma is specific for polymerase Ib. 3. The curve of UMP incorporation in vitro, with and without factors gamma or delta suggests that they are initiation factors. This conclusion is reinforced by the analysis of simultaneous incorporation of [gamma-32P]ATP and [3H]UMP in the RNAs synthesized in vitro. 4. Although the level of factor delta is independent of auxin treatment, that of factor gamma is doubled in auxin-treated roots. These results suggest that factor gamma is an auxin-induced protein that modulates the specific activity of the nucleolar RNA polymerase. 5. A general model of the mode of action of auxins at the molecular level is proposed. It integrates into a unified scheme the above results as well as those obtained by other workers.