DNA structural variation affects complex formation and promoter melting in ribosomal RNA transcription.

Eukaryotic ribosomal RNA promoters exhibit an unusual conservation of non-canonical DNA structure (curvature, twist angle and duplex stability) despite a lack of primary sequence conservation. This raises the possibility that rRNA transcription factors might utilize structural anomalies in their sequence recognition process. We have analyzed in detail the interaction of the polymerase I transcription factor TIF-IB from Acanthmoeba castellanii with the CORE promoter. TIF-IB interacts primarily with the minor groove of the promoter. By correlating the effects on transcription and on DNA structure of promoter point mutations, we show that the TIF-IB interaction is strongly inhibited by increases in minor groove width. This suggests that a particular DNA structure is required for interaction with the transcription factor. In addition, TIF-IB induces a small bend in the promoter upon binding. Modeling of this bend reveals that it requires an additional narrowing of the minor groove, which would favor binding to mutants with narrower grooves. We also discuss how this narrowing would induce a small destabilization of the helix upstream of the transcription start site. Telestability predicts this would result in destabilization of the sequence that melts during initiation, suggesting that TIF-IB may have a role in stimulating melting.

Modeling of DNA local parameters predicts encrypted architectural motifs in Xenopus laevis ribosomal gene promoter.

We have modeled local DNA sequence parameters to search for DNA architectural motifs involved in transcription regulation and promotion within the Xenopus laevis ribosomal gene promoter and the intergenic spacer (IGS) sequences. The IGS was found to be shaped into distinct topological domains. First, intrinsic bends split the IGS into domains of common but different helical features. Local parameters at inter-domain junctions exhibit a high variability with respect to intrinsic curvature, bendability and thermal stability. Secondly, the repeated sequence blocks of the IGS exhibit right-handed supercoiled structures which could be related to their enhancer properties. Thirdly, the gene promoter presents both inherent curvature and minor groove narrowing which may be viewed as motifs of a structural code for protein recognition and binding. Such pre-existing deformations could simply be remodeled during the binding of the transcription complex. Alternatively, these deformations could pre-shape the promoter in such a way that further remodeling is facilitated. Mutations shown to abolish promoter curvature as well as intrinsic minor groove narrowing, in a variant which maintained full transcriptional activity, bring circumstantial evidence for structurally-preorganized motifs in relation to transcription regulation and promotion. Using well documented X. laevis rDNA regulatory sequences we showed that computer modeling may be of invaluable assistance in assessing encrypted architectural motifs. The evidence of these DNA topological motifs with respect to the concept of structural code is discussed.

Structure-function relationships in replication origins of the yeast Saccharomyces cerevisiae: higher-order structural organization of DNA in regions flanking the ARS consensus sequence.

In order to better understand the involvement of the DNA molecule in the replication initiation process we have characterized the structure of the DNA at Autonomously Replicating Sequences (ARSs) in Saccharomyces cerevisiae. Using a new method for anti-bent DNA analysis, which allowed us to take into account the bending contribution of each successive base plate, we have investigated the higher-order structural organization of the DNA in the region which immediately surrounds the ARS consensus sequence (ACS). We have identified left- and right-handed anti-bent DNAs which flank this consensus sequence. The data show that this organization correlates with an active ACS. Analysis of the minimum nucleotide sequence providing ARS function to plasmids reveals an example where the critical nucleotides are restricted to the ACS and the right-handed anti-bent DNA domain, although most of the origins considered contained both left- and right-handed anti-bent DNAs. Moreover, mutational analysis shows that the right-handed form is necessary in order to sustain a specific DNA conformation which is correlated with the level of plasmid maintenance. A model for the role of these individual structural components of the yeast replication origin is presented. We discuss the possible role of the right-handed anti-bent DNA domain, in conjunction with the ACS, in the process of replication initiation, and potentialities offered by the combination of left- and right-handed structural components in origin function.

A sequence based computational identification of a Drosophila developmentally regulated TATA-less RNA polymerase II promoter and its experimental validation.

Many RNA polymerase II promoters lack the characteristic TATA box sequence located -25/-30 nucleotides upstream from the transcription start. In Drosophila, half of the promoters identified so far are TATA-deficient. The yemanuclein-alpha gene whose promoter activity is restricted to oogenesis, falls in this class. A number of upstream and downstream promoter elements have been identified for some TATA-less promoters. The yem-alpha promoter contains none of the consensus elements identified so far. Our work was based on the assumption that the physical parameters of the DNA could be used to predict the location of the yem-alpha promoter. A sequence based computational analysis allowed us to determine the characteristic changes of DNA curvature and helix stability in the presumptive regulatory region. Our experimental data were in good agreement with the computational analysis. We have started to investigate the general value of this approach by analyzing other promoters.

Common DNA structural features exhibited by eukaryotic ribosomal gene promoters.

Nucleotide sequences of DNA regions containing eukaryotic ribosomal promoters were analysed using strategies designed to reveal sequence-directed structural features. DNA curvature, duplex stability and pattern of twist angle variation were studied by computer modelling. Although ribosomal promoters are known to lack sequence homology (unless very closely related species are considered), investigation of these structural characteristics uncovered striking homologies in all the taxonomic groups examined so far. This wide conservation of DNA structures, while DNA sequence is not conserved, suggests that the determined structures are fundamental for ribosomal promoter function. Moreover, this result agrees well with the recent observations showing that RNA polymerase I transcription factors have not evolved as intensively as previously suspected.

Promoter structure and intron-exon organization of a scorpion alpha-toxin gene.

The Androctonus australis scorpion venom contains alpha-toxins for which the complementary DNAs have been cloned [Bougis et al. (1989) J. Biol. Chem. 264, 19259-19265], targeting with high affinity the voltage-sensitive sodium channel. From a genomic library made of this species of scorpion, we have cloned and characterized the gene encoding the toxin AaH I'. The gene transcriptional unit is 793 base pairs long, and the gene has a single intron of 425 base pairs located near the end of the signal peptide of the toxin precursor. The transcription initiation site was determined by primer extension and corresponded to the nucleotide sequence AACAA. Upstream, a promoter region has been identified with positive acting sequence elements at consensus positions, such as a CCAAT box and a TATA box. In addition, putative elements for binding the transcriptional factors MAT-alpha 2, Pit-1, and IEF1 are also present. Analysis of DNA curvature by computer modeling revealed a strong bending centered around the transcription initiation site of the gene. The bending angle (61 degrees) estimated experimentally using polyacrylamide gel electrophoresis correlates well with the value predicted by computer modeling (66 degrees). Other minor deflections of the helix axis cooperate for an overall curvature of nearly 90 degrees, which is significantly stronger than similar structures already reported in eukaryotic cells. It is worth noting that the grooves relative to the CCAAT box and the TATA box lie along the inside of the DNA curve. This observation is in agreement with the previously reported correlation between DNA bending and promoter function.

Long-range organization and sequence-directed curvature of Xenopus laevis satellite 1 DNA.

We have investigated the long-range organization and the intrinsic curvature of satellite 1 DNA, an unusual tandemly-repeated DNA family of Xenopus laevis presenting sequence homologies to SINEs. PFGE was used in combination with frequent-cutter restriction enzymes not likely to cut within satellite 1 DNA and revealed that almost all the repeating units are tandemly organized to form large arrays (200 kb to 2 Mb) that are marked by restriction length polymorphism and contain intra-array domains of sequence variation. Besides that, we have analysed the secondary structure of satellite 1 DNA by computer modelling. Theoretical maps of curvature obtained from three independent models of DNA bending (the dinucleotide wedge model of Trifonov, the junction model of Crothers and the model of de Santis) showed that satellite 1 DNA is intrinsically curved and these results were confirmed experimentally by polyacrylamide gel electrophoresis. Moreover, we observed that this bending element is highly conserved among all the members of the satellite 1 DNA family that are accessible to analysis. A potential genetic role for satellite 1 DNA based on this unusual structural feature is discussed.

Size variation of rDNA clusters in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe.

The higher-order organization of rRNA genes was investigated in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. We used pulsed-field gel electrophoresis (PFGE) in combination with frequent cutter endonucleases having no recognition sites within rDNA repeating units to characterize tandem arrays of ribosomal genes in these two species. Large variations in rDNA cluster length were detected in various S. cerevisiae and S. pombe strains commonly used as PFGE molecular weight markers. This wide range of variability implies that the sizes currently assessed for chromosomes bearing rRNA genes in these organisms are unreliable since they may vary within strains by several hundreds of kilobase pairs, depending on the size of the tandem arrays of rRNA genes. Consequently, there is now a lack of reliable PFGE size standards between 1.6 Mb and 4.5 Mb, even when established yeast strains with calibrated chromosomes are used.

Molecular dissection of a specific nuclear domain: the chromatin region of the ribosomal gene cluster in Xenopus laevis.

Molecular dissection of the nuclear domain corresponding to the ribosomal chromatin cluster was investigated. The experimental scheme was based on the ability of restriction enzymes to digest the whole genome without affecting this region (several megabases in length). Such a strategy involved the judicious choice of restriction enzymes, which is possible in Xenopus laevis, where the rDNA sequence is known and the repeated units are organized into one unique cluster. SalI, XhoI, and EcoRV digestion produced frequent cutting of the genome leaving the ribosomal cluster intact. Isolation of the rDNA cluster was confirmed by separation of the digested DNA by pulsed-field electrophoresis. When applied to purified nuclei, this approach allowed the isolation of the ribosomal chromatin cluster under very mild conditions: no cleavages (either enzymatic or mechanical) were detectable. Since the purification scheme depends only on the DNA sequence outside of the rDNA cluster, it permits the obtention of this domain in different functional states. Electron microscopic analysis demonstrated that the domain organization is substantially preserved and maintains its looped organization (the size and the full number of loops were preserved). This purification scheme provides a powerful tool for studying the structure-function relationships within the ribosomal nuclear domain.

Supercoiled loop organization of genomic DNA: a close relationship between loop domains, expression units, and replicon organization in rDNA from Xenopus laevis.

Analysis of the organization of a specific chromosomal gene, the gene for rRNA in Xenopus laevis, has evidenced a close relationship between loop organization, replication organization, and expression units. The nontranscribed spacer appears to be involved in all three levels of organization. Furthermore the replication origin region appears to be involved in nuclear matrix anchorage and is closely related to the 40 S transcription promoter. This organization suggests how expression domains may be regulated and how this functional organization may be transmitted to daughter cells after DNA replication, thus allowing selected expression patterns not to be lost during development.

Variations in accessibility of DNA during traumatic regeneration by Owenia fusiformis (polychaete annelid).

One of the earliest events observed in the 'dedifferentiation phase' of traumatic regeneration is an in vivo increase in transcription. In order to see whether that increase corresponds to a variation at the level of genetic material, we prepared chromatin from normal and regenerating animals (12 h after amputation) for transcription in vitro by E. coli RNA polymerase. The template activity of regenerating chromatin was 2.1-fold higher than normal under standard conditions and showed an altered response to variation of the ionic strength of the medium. The accessibility of DNA in the chromatin was tested using mild digestion by DNAase II. Almost twice as much DNA was solubilised from regenerating chromatin as from normal chromatin. These data were correlated with morphological changes (decondensation) observed by electron microscopy.

Fluctuations of adenylate cyclase activity during anterior regeneration in Owenia fusiformis (Polychaete Annelid).

Biochemical assays of adenylate cyclase activity were performed during the early phases of regeneration in Owenia fusiformis (Polychaete Annelid). The results indicate the existence of a strong stimulation in an early phase following trauma. This stimulation is then followed by periodic fluctuations exhibiting a diurnal rhythm correlated with the cell cycle. Adenylate cyclase activity is also shown to be neurotransmitter-dependent. In this paper it is proposed that neurotransmitters might participate in the regulation of cyclic AMP formation, by means of adenylate cyclase acting on target blastema cells, undergoing the cell cycle.

DNA synthesis during the first stages of anterior regeneration in the polychaete annelid Owenia fusiformis (dedifferentiation and early phases of differentiation).

We have analysed DNA synthesis in early phases of regeneration in a marine Polychaete Annelid, Owenia fusiformis. The length and efficiency of the prereplicative phase was found to vary with the diurnal rhythm of activity of the animal; that is, it depends on the initial state of the cell population at the moment of the onset of proliferative stimulatin. When animals were operated on at 12 a.m., the duration of the prereplicative phase of the first cells stimulated to proliferate was found to be 12 h. The remaining cells entered the S-phase progressively in waves until the 3rd day following amputation when nearly 100% of the blastema cells were stimulated. At that time the cell-cycles of these dividing cells were found to be highly synchronized. Blastema differentiation takes place on the 4th day and is initiated by stomodeum formation. During the differentiation phase, DNA synthesis is restricted to small areas of the regenerating part. The system described is viewed as a new instrument for investigating the control of the cell cycle in synchronized and subsequently differentiating tissue cells.

[Daily variations of the level of neural secretions in Owenia fusiformis. Persistance of a daily rhythm in cultured tissues]. / Variations journalières du taux des sécrétions nerveuses chez Owenia fusiformis. Persistance d'un rhythme journalier dans les tissus en culture

A quantitative study of elementary granules in neuron axons of the nervous chain of Owenia fusiformis (Annelid polychaete) shows that daily variations of the number of secretory granules occur in unamputated animals and in ventral tissue cultures. These variations are correlated with the thythm of the cell cycle.