A small sequence in domain v of the mitochondrial large ribosomal RNA restores Drosophila melanogaster pole cell determination in uv-irradiated embryos.

The mechanism by which the mitochondrial large rRNA is involved in the restoration of the pole cell-forming ability in Drosophila embryos is still unknown. We identified a 15-ribonucleotide sequence which is conserved from the protobacterium Wolbachia to the higher eukaryotes in domain V of the mitochondrial large rRNA. This short sequence is sufficient to restore pole cell determination in UV-irradiated Drosophila embryos.

The HIV Tat protein affects processing of ribosomal RNA precursor.

Background: Inside the cell, the HIV Tat protein is mainly found in the nucleus and nucleolus. The nucleolus, the site of ribosome biogenesis, is a highly organized, non-membrane-bound sub-compartment where proteins with a high affinity for nucleolar components are found. While it is well known that Tat accumulates in the nucleolus via a specific nucleolar targeting sequence, its function in this compartment it still unknown.

The HIV-Tat protein induces chromosome number aberrations by affecting mitosis.

To analyze the effects of the HIV-Tat-tubulin interaction, we microinjected HIV-Tat purified protein into Drosophila syncytial embryos. Following the Tat injection, altered timing of the cortical nuclear cycles was observed; specifically, the period between the nuclear envelope breakdown and anaphase initiation was lengthened as was the period between anaphase initiation and the formation of the next nuclear envelope. These two periods correspond to kinetochore alignment at metaphase and to mitosis exit, respectively.

A Drosophila model of HIV-Tat-related pathogenicity.

To analyze the mechanism of Tat-mediated HIV pathogenicity, we produced a Drosophila melanogaster strain transgenic for HIV-tat gene and induced the expression of the protein during Drosophila development. By in vitro and in vivo experiments, we demonstrated that Tat specifically binds to tubulin via the MAP-binding domain of tubulin, and that this interaction delays the polymerization of tubulin and induces a premature stop to microtubule-dependent cytoplasmic streaming. The delay in the polymerization of microtubules, the tracks for the transport of the axes determinants, alters the positioning of the dorso-ventral axis as shown by the mislocalization of Gurken and Kinesin in oocyte of Drosophila after Tat induction.

Analysis of HIV-1 Tat effects in Xenopus laevis embryos.

Tat is one of the regulatory proteins of the HIV-1 virus. To date, besides the transactivation activity, a myriad of effects exerted by HIV-1 Tat on cellular and viral genes have been observed. The present study investigated the in vivo effects of HIV-1 Tat protein in the Xenopus embryo.

Measurement of the range of HIV-LTR transactivating activity of HIV in vitro.

Tat is known to act both in a 'cell autonomous' and in 'cell non-autonomous' manner, i.e. Tat can function as a powerful transcription factor both inside the cell in which it is expressed and also in cells not expressing Tat.

Interactions among the bHLH domains of the proteins encoded by the Enhancer of split and achaete-scute gene complexes of Drosophila.

The Enhancer of split and achaete-scute gene complexes [E(spl)-C and AS-C] encode helix-loop-helix proteins required for neurogenesis in Drosophila. Using a heterologous bacterial system, we show that (i) the bHLH domains of the proteins encoded by the two gene complexes differ in their ability to form homo- and/or heterodimers; (ii) the bHLH domains of the E(spl)-C proteins m5, m7 and m8 interact with the bHLH domains of the Ac and Sc proteins. These bHLH domains form an interaction network which may represent the molecular mechanism whereby the competent state of the proneural cells is maintained until the terminal determination to neuroblast occurs.

A novel approach to protein-protein interaction: complex formation between the p53 tumor suppressor and the HIV Tat proteins.

By using a novel genetic approach, based on the properties of lambda cl repressor, we demonstrate that the HIV-1 Tat protein specifically interacts with the human p53 protein via the p53 O2 dimerization domain. By random and site-specific mutagenesis, we also identify the residues in Tat and O2 peptides which are involved in this interaction. Two alternative biological consequences are expected to result from Tat-p53 interaction: (i) Tat-O2 interaction inactivates p53 regulation function, thus producing cell transformation; (ii) Tat-O2 interaction favours the formation of p53 dimers, thus leading the cell towards apoptosis.

Genetic tests to reveal TAT homodimer formation and select TAT homodimer inhibitor.

The HIV Tat protein is essential for productive infection and is a potent activator of viral gene expression. By constructing a genetic fusion between the amino-terminal DNA-binding domain of the lambda repressor (as a reporter for dimerization) and Tat, we show that Tat forms dimers in vivo. By deletion analysis and site-directed mutagenesis, we show that (i) the peptide encoded by exon-1 of Tat is sufficient to promote dimerization and (ii) cys37 is essential for homo-dimerization of Tat protein.

pR plasmid replication provides evidence that single-stranded DNA induces the SOS system in vivo.

Evidence is presented that the pR bat gene is essential for plasmid replication and for spontaneous induction of the SOS response in Escherichia coli. Mutations preventing single-stranded DNA production, needed for pR plasmid replication, also prevent the induction of the SOS system. The following experimental design was used.

The uvp1 gene of plasmid pR cooperates with mucAB genes in the DNA repair process.

We show that a DNA fragment that contains the uvp1 gene of the plasmid pR directs the synthesis in Escherichia coli minicells of a protein of apparent molecular weight 20 kDa. Inspection of the nucleotide sequence of the region reveals an open reading frame that has the capacity to encode a protein of 198 amino acids. The uvp1 gene product has been found, in two different systems, to enhance the recombinational activity of E.

The pR plasmid: a tool for studying DNA repair and mutagenesis in prokaryotic and eukaryotic cells.

The pR plasmid, a derivative of R46 plasmid, offers the possibility to have an experimental approach to three important problems related to UV repair and mutagenesis. By using this plasmid we were able to show: a) the pR mucAB genes need the cooperation of uvpl gene product to carry out their UV repair function; b) the expression of mucAB genes is regulated not only by lexA gene, but by a gene localized in the rep region of pR itself. This gene acts as an antirepressor of lexA; c) mammalian cells show an enhanced resistance to UV light when transformed by pR plasmid carrying the mucAB genes.

The rep region of pR plasmid regulates the expression of SOS system.

By using an artificial hybrid between phage lambda and the pR plasmid, we have shown that the rep region of the pR plasmid encodes a function which regulates the expression of the muc genes (plasmid genes that are under the negative control of lexA and responsible for an increased rate of spontaneous mutagenesis and resistance to UV and chemicals). Expression of the muc genes were monitored by a fusion between the muc promoter and the lacZ structural gene. When E.

The pR plasmid: a tool for discriminating between DNA lesions induced by different types of cytotoxic agents in cultured mammalian cells.

The LA-D cells, obtained by cotransformation of LTA mouse cells (tk- aprt-) with pR plasmid and with tk gene as selective marker, are significantly more resistant to UV light and 4-nitroquinoline-N-1-oxide than LTA control cells. In this work, we report that the LA-D cells exhibit different degrees of response to various DNA-damaging agents: wild-type survival to mitomycin, increased sensitivity to bleomycin, cis-diamminedichloroplatinum and N-methyl-N'-nitro-N-nitrosoguanidine. The pR plasmid could, therefore, play an important role in the DNA-repair mechanisms that modulate the cytotoxic effect of the DNA-inhibitory agents.

Cellular response to DNA damage is enhanced by the pR plasmid in mouse cells and in Escherichia coli.

The pR plasmid, which enhances the survival of Escherichia coli C600 exposed to UV light by induction of the SOS regulatory mechanism, showed the same effect when it transformed mouse LTA cells (tk-, aprt-). With Tn5 insertion mutagenesis which inactivates UV functions in the pR plasmid, we recognized two different regions of the plasmid, uvp1 and uvp2. These pR UVR- mutants exhibited the same effect in LTA transformed cells, demonstrating that resistance to UV light, carried by the pR plasmid, was really due to the expression of these two regions, which were also in the mouse cells.

The pR UV+ plasmid, transfected into mammalian cells, enhances their UV survival.

It has been recently reported that the pR plasmid enhances the UV survival in E.coli c600. In order to test whether this function may be expressed also in mammalian cells, LTA (tk- aprt-) mouse cells were cotransformed with pR plasmid DNA and ptk1 plasmid as selectable marker.

Identification of a protein coded by pR plasmid and involved in SOS repair in E. coli.

The TP120 plasmid is known to determine enhanced UV survival in E. coli wild type an uvrB and PolA mutants but not in RecA mutant. In order to analyze the function involved in the SOS repair, we have constructed a new plasmid named pR derived by cleavage of TP120 with Hind III endonuclease.

Peculiar mosaicism 47,XYY/48,XYYY/49,XYYYY in man.

The chromosome analysis in a 9 years-old boy showed the presence of three cell lines : 47,XYY (28%) ; 48,XYYY (68%); 49,XYYYY (4%). Since the most frequent cells bear three Y chromosomes and the karyotype of the propositu's father is normal, it is suggest that the propositus arose from an YYY sperm and that the observed mosaicism originated from a subsequent postzygotic non-disjunction.

Partial deletion of the X chromosome in gonadal dysgenesis 46,X,del(X)(p22) identified by BudR treatment.

In this report we describe a deletion of the short arm of the X chromosome in a 16-year-old female with gonadal dysgenesis. The breakpoint was localized by BUdR treatment and acridine orange staining in region 2, band 2. Of the examined cells, 3% showed an early replication of the deleted X chromosome.