Raymond Miassod

Studies of an 800-kilobase DNA stretch of the Drosophila X chromosome: comapping of a subclass of scaffold-attached regions with sequences able to replicate autonomously in Saccharomyces cerevisiae.

We have previously mapped scaffold-attached regions (SARs) on an 800-kilobase DNA walk from the Drosophila X chromosome. We have also previously shown that the strength of binding, i.e., the ability of SARs to bind to all nuclear scaffolds or only to a fraction of them varied from one SAR to another one. In the present study, 71 of the 85 subfragments that bind scaffolds and 38 fragments that do not bind scaffolds were tested for their ability to promote autonomous replicating sequence (ARS) activity in Saccharomyces cerevisiae. Sixteen SAR-containing fragments from the chromosome walk were also examined for association to yeast nuclear scaffolds in vitro. All identified ARSs (a total of 27) were present on SAR-containing fragments, except two, which were adjacent to SARs. There is thus a correlation between ARS and SAR activities, and this correlation defines a SAR subclass. Moreover, the presence of an ARS on a DNA fragment appeared to be highly correlated with the strength of binding. The binding activity was highly conserved from Drosophila melanogaster to yeast. These data suggest that Drosophila DNA sequences responsible for binding to components of the nuclear scaffold from either D. melanogaster or yeast may be involved in the process of heterologous extrachromosomal replication in yeasts.

Mutations in ccf, a novel Drosophila gene encoding a chromosomal factor, affect progression through mitosis and interact with Pc‐G mutations

We report herein the isolation of ccf, a new gene located in region 82E and essential for Drosophila development. This gene, expressed throughout development, encodes a novel product of 68 kDa which is found in the nucleus during interphase and labels, in a novel pattern, centrosomes and chromosome arms during mitosis. Mutations in ccf give rise to late larvae with small imaginal discs and to adults showing appendages of reduced size, consistent with CCF involvement in cell proliferation. Neuroblast squash analyses show that CCF is required for proper condensation of mitotic chromosomes and, therefore, for progression through mitosis. Furthermore, we observe that adult ccf mutants as well as animals overexpressing CCF during larval stages exhibit homeotic transformations. We also find that mutations in the Pc‐G genes Polycomb, polyhomeotic and Enhancer of zeste are enhanced by ccf mutations. Finally, we show that the CCF protein binds to specific sites on polytene chromosomes, many of which are shared with the Posterior sex combs Pc‐G protein. Together, these results suggest a role for the CCF protein in the maintenance of chromosome structure during mitosis and interphase.

Peroxidases associated with lentil root ribosomes

Abstract Three peroxidases are associated with the ribosomes of lentil roots. During cellular fractionation, the three haemoproteins are only present on these particles and not in the other fractions. The three peroxidases were isolated and obtained in a highly purified form. Their spectral characteristics and their molecular weights were determined. One is slightly acidic at pH 6·8, whereas the other two are slightly basic at this same pH. The incorporation of a 14 C-amino acid into these haemoproteins has enabled us to show that the basic peroxidases turn over rapidly whereas the acid peroxidase is very stable. Treatment of the roots with IAA considerably stimulates the de novo biosynthesis of the two basic peroxidases but not that of the acid peroxidase.

dlarp, a new candidate Hox target in Drosophila whose orthologue in mouse is expressed at sites of epithelium/mesenchymal interactions.

Hox complex genes are key developmental regulators highly conserved throughout evolution. They encode transcription factors that initiate genetic programs of diversified morphogenesis along the anteroposterior embryonic axis. We report the characterization of the novel Drosophila Hox target gene dlarp, isolated from a further screen of a previously described library of genomic DNA fragments associated in vivo with Ultrabithorax proteins. The dlarp spatio‐temporal pattern of transcription in wild‐type and homeotic mutant embryos is consistent with a positive regulation by Sex combs reduced and Ultrabithorax in the parasegment 2 ectoderm and the abdominal mesoderm, respectively. The teashirt gene product, thought to act in concert with Hox proteins, is also required for the transcriptional control of this target. Search in databases revealed that dlarp has been highly conserved during evolution. The embryonic expression pattern of the mouse orthologue does not support a function downstream of Hox proteins. It is mainly transcribed in neural structures and in developing organs characterized by epithelial‐mesenchymal interactions. Dev Dyn 2000;218:401–413.

Supragenic loop organization: mapping in Drosophila embryos, of scaffold-associated regions on a 800 kilobase DNA continuum cloned from the 14B-15B first chromosome region.

The supragenic loop organization of the Drosophila genome was investigated on a 800 kilobase (kb) DNA continuum from the 14B-15B first chromosome region. Nuclear scaffolds from 0-18 hr embryos were prepared with Laemmlis low-salt, detergent procedure and digested with restriction enzymes. Scaffold-associated regions (SARs) were mapped by probing Southern transfers of total, scaffold-associated and free DNA with a set of 70 recombinant phages overlapping the investigated genomic region. In all, 85 restriction fragments showed association to scaffolds. 12 of them were present in the majority of scaffolds. They bore strong SARs organizing the DNA molecule as consecutive loops with sizes ranging from 15 to 115 kb. 44 were present in only a fraction of scaffolds. They contained weak SARs subdividing the basic loops into smaller ones. 29 additional restriction fragments were present in a very small fraction of scaffolds. The position of SARs with respect to transcribed regions was investigated. Strong SARs appeared to be located on untranscribed DNA and to frame transcription units. In contrast, at least some weak SARs were shown to comap with transcribed regions or to reside within characterized transcription units. Statistical analyses established that strong and weak SARs were periodically positioned on the DNA continuum and that there was a potential contact point between scaffolds and the DNA continuum every 11 kb, or multiples thereof. Implications for SAR role(s) are discussed.

Ribosomal cistrons in higher plant cells. I. A definitive scheme for the maturation pathway of the primary transcriptional product of ribosomal cistrons in Acer pseudoplatanus L. cells.

1. Four precursor ribosomal RNAs have been previously characterized in cultured sycamore cells (Acer pseudoplatanus L.). The present paper reports a study of the two smallest ones. 2. (Me-3H)-labelling of the precursor and mature rRNAs has been submitted to kinetic analysis. From pulse-chase experiments it is concluded that the 27S and 20S precursor rRNAs behave like intermediate molecules in the maturation of the 42S and 36S precursor rRNAs into the 26S and 17S components. They are direct precursors to the cytoplasmic and mature rRNAs. 3. Base-composition analyses have been performed on carefully purified molecules. They show that the 27S and 20S precursor rRNAs arise from a single cleavage of the 36S precursor rRNA. In addition, they strongly suggest that the 27S and 20S precursor rRNAs can be taken as specific precursors to the 26S and 17S rRNAs, respectively. These results are reinforced by DNA - RNA hybridization experiments, the measured values being in good agreement with the theoretical ones expected from a specific relationship between the four smallest molecules. Base-compositions of the conserved and non-conserved nucleotide sequences of the primary transcriptional product have been estimated or calculated. 4. On the basis of the present results and those previously reported, a scheme for the biosynthesis of ribosomal RNA in sycamore cells is proposed.

Contribution a l'etude d'acides ribonucleiques a marquage rapide de la racine de lentille: Isolement et caracterisation, action d'une hormone sur leur synthese et sur celle de peroxydases

Abstract Chromatography on methylated albumin columns of RNAs from Lentil roots, shows the presence of rapidly labelled RNAs tenaciously bound onto the column and which cannot be eluted by high NaCl molarities. Elution at 35°, with sodium dodecyl sulfate, gives two peaks (α and β). At 70°, a third elution peak (γ) is obtained. For short labelling periods, RNAs of the α peak are uridine rich (UMP/AMP approx. 1.2), heterodisperse and may have an important percentage of high-molecular-weight species. RNAs from the β peak are adenine rich (UMP/AMP approx. 0.4). Their modal sedimentation constant is about 12 S. The mean life of the uridine-rich RNAs of the α peak is lower than 1 h; that of the RNAs from the β peak is higher than 2 h. RNAs from α peak are essentially, or exclusively, of nuclear origin. On the other hand, polysomic messenger RNAs are present in the β peak. A close similitude exists between uridine-rich RNAs described in animal cells and the nuclear rapidly labelled RNAs of Lentil roots contained in the α peak. Short treatment (45 min) of Lentil roots with indoleacetic acid increases the amount of nuclear uridine-rich RNAs. A prolonged treatment (5 h) causes an increase in various rapidly labelled RNAs, probably polysomic messenger RNAs. Treatment of excised roots with indoleacetic acid leads, after a lag period of about 5 h, to the synthesis of two peroxydases associated with the ribosomes. The mean life of their messenger RNAs is about 2.5 h.

nessy, an evolutionary conserved gene controlled by Hox proteins during Drosophila embryogenesis

From a library of DNA fragments associated with Ultrabithorax protein in vivo, we have isolated nessy, a new Drosophila gene that encodes a putative transmembrane protein conserved in evolution from Caenorhabditis elegans, to human. Zygotic expression occurs transiently in mesectodermal cells at gastrulation, proceeds in mesoderm and endoderm lineages during germ band movements and becomes then restricted to anterior and posterior domains in the visceral mesoderm. The Hox proteins Ultrabithorax, Antennapedia and AbdominalA are likely acting simultaneously to repress nessy in the other parts of the visceral mesoderm.

Ribosomal cistrons in higher plant cells: II. Sequence homology between the two mature rRNAs of sycamore cells and intracistronic reiteration. A DNA · rRNA hybridization study

1. Uniformly labelled rRNA of sycamore cells has been annealed with homologous DNA. The fractions of DNA complementary to the 17S, or 26S, or 17S + 26S rRNAs are found to be 0.19%, 0.15% and 0.23%. They are not in the ratio of the molecular weight values (0.8, 1.2 and 2 - 10(6), respectively for the 17S, 26S and 17S + 26S rRNAs). This result is compatible with the large hybridization competition observed between the two rRNAs (53 and 72%) and with the shift-down of saturation curves when DNA is presaturated with unlabelled rRNA before the incubation with the other labelled rRNA. 2. Under the selected experimental procedure, the DNA - rRNA hybrids formed appear to be specific. Since there is an equal number of structural genes for the 17S and 26S rRNAs, these results mean the occurrence of a great sequence homology, strictly restricted to the two rRNAs. Homologous and specific sequences have been estimated to 0.1 and 0.7, or 0.85 and 0.35 million daltons, respectively in the 17S or 26S structural genes. 3. From the calculated lengths of homologous sequences, an intracistronic reiteration of some ribosomal sequences can be deduced. This internal reiteration is directly evidenced by the complex pattern of DNA - rRNA annealing curves. As demonstrated by base-composition analysis, the internal reiteration is heterogeneous and concerns both the homologous and specific sequences. In addition, the DNA saturation values allow the calculation of 4000 copies for the ribosomal cistron in the whole sycamore genome.

Scaffold-associated regions and repeated or cross-hybridizing sequences on an 800 kilobase DNA stretch of the Drosophila X chromosome

Summry— DNA fragments binding to the scaffold (SARs) have been previously mapped on an 800‐kb DNA fragment from the Drosophila × chromosome. Here we have examined whether these 86 DNA fragments bear sequences repeated in the Drosophila genome and/or cross‐hybridizing sequences. Twenty‐two middle‐repeated sequences were localized by hybridizing Southern transfers of representative cloned DNA with total genomic DNA and, reciprocally, by hybridizing Southern transfers of total genomic DNA with this cloned DNA. Seventy‐nine out of the 86 SAR‐containing fragments appeared to be distinct from these 22 repeated motifs. Therefore, SARs, in their vast majority, are not members of middle‐repeated sequence families. However, the 22 middle‐repeated sequences were shown to be significantly located in the near vicinity of SARs. Hybridizations were also performed between SAR‐containing DNA fragments, either at a high or at a low stringency. At a high stringency, the 86 SAR‐containing DNA fragments did not cross‐hybridize. However, at a low stringency, a complex hybridization network was evidenced. These nucleotide sequence similitudes support the idea that the various SARs may play common roles.