Search PubMed⌕ Search

Biomedical subjects

R Sandaltzopoulos

Publications and source records attributed to R Sandaltzopoulos.

18 recordsLinked to original sources
PubMed2004

An algorithm for the study of DNA sequence evolution based on the genetic code.

Recent studies of the quantum-mechanical processes in the DNA molecule have seriously challenged the principle that mutations occur randomly. The proton tunneling mechanism causes tautomeric transitions in base pairs resulting in mutations during DNA replication. The meticulous study of the quantum-mechanical phenomena in DNA may reveal that the process of mutagenesis is not completely random. We are still far away from a complete quantum-mechanical model of DNA sequence mutagenesis because of the complexity of the processes and the complex three-dimensional structure of the molecule. In this paper we have developed a quantum-mechanical description of DNA evolution and, following its outline, we have constructed a classical model for DNA evolution assuming that some aspects of the quantum-mechanical processes have influenced the determination of the genetic code. Conversely, our model assumes that the genetic code provides information about the quantum-mechanical mechanisms of mutagenesis, as the current code is the product of an evolutionary process that tries to minimize the spurious consequences of mutagenesis. Based on this model we develop an algorithm that can be used to study the accumulation of mutations in a DNA sequence. The algorithm has a user-friendly interface and the user can change key parameters in order to study relevant hypotheses.

Algorithms↗
PubMed2001

Dual functions of largest NURF subunit NURF301 in nucleosome sliding and transcription factor interactions.

NURF is an ISWI complex of four proteins that uses the energy of ATP hydrolysis to catalyze nucleosome sliding. Three NURF components have been identified previously. We have cloned cDNA encoding the largest NURF subunit, revealing a 301 kDa polypeptide (NURF301) that shares structural motifs with ACF1. We have reconstituted full and partial NURF complexes from recombinant proteins and show that NURF301 and the ISWI ATPase are necessary and sufficient for accurate and efficient nucleosome sliding. An HMGA/HMGI(Y)-like domain of NURF301 that facilitates nucleosome sliding indicates the importance of DNA conformational changes in the sliding mechanism. NURF301 also shows interactions with sequence-specific transcription factors, providing a basis for targeted recruitment of the NURF complex to specific genes.

Amino Acid Motifs↗
PubMed1999

ATP-dependent histone octamer sliding mediated by the chromatin remodeling complex NURF.

Drosophila NURF is an ATP-dependent chromatin remodeling complex that contains ISWI, a member of the SWI2/SNF2 family of ATPases. We demonstrate that NURF catalyzes the bidirectional redistribution of mononucleosomes reconstituted on hsp70 promoter DNA. In the presence of NURF, nucleosomes adopt one predominant position from an ensemble of possible locations within minutes. Movements occur in cis, with no transfer to competing DNA. Migrating intermediates trapped by Exo III digestion reveal progressive nucleosome motion in increments of several base pairs. All four core histones are retained quantitatively during this process, indicating that the general integrity of the histone octamer is maintained. We suggest that NURF remodels nucleosomes by transiently decreasing the activation energy for short-range sliding of the histone octamer.

Adenosine Triphosphatases↗
PubMed1998

Inorganic pyrophosphatase is a component of the Drosophila nucleosome remodeling factor complex.

The Drosophila nucleosome remodeling factor (NURF) is a protein complex consisting of four polypeptides that facilitates the perturbation of chromatin structure in vitro in an ATP-dependent manner. The 140-kD NURF subunit, imitation switch (ISWI), is related to the SWI2/SNF2 ATPase. Another subunit, NURF-55, is a 55-kD WD repeat protein homologous to the human retinoblastoma-associated protein RbAp48. Here, we report the cloning and characterization of the smallest (38 kD) component of NURF. NURF-38 is strikingly homologous to known inorganic pyrophosphatases. Both recombinant NURF-38 alone and the purified NURF complex are shown to have inorganic pyrophosphatase activity. Inhibition of the pyrophosphatase activity of NURF with sodium fluoride has no significant effect on chromatin remodeling, indicating that these two activities may be biochemically uncoupled. Our results suggest that NURF-38 may serve a structural or regulatory role in the complex. Alternatively, because accumulation of unhydrolyzed pyrophosphate during nucleotide incorporation inhibits polymerization, NURF may also have been adapted to deliver pyrophosphatase to chromatin to assist in replication or transcription by efficient removal of the inhibitory metabolite.

Amino Acid Sequence↗
PubMed1998

Prothymosin alpha modulates the interaction of histone H1 with chromatin.

Prothymosin alpha (ProTalpha) is an abundant acidic nuclear protein that may be involved in cell proliferation. In our search for its cellular partners, we have recently found that ProTalpha binds to linker histone H1. We now provide further evidence for the physiological relevance of this interaction by immunoisolation of a histone H1-ProTalpha complex from NIH 3T3 cell extracts. A detailed analysis of the interaction between the two proteins suggests contacts between the acidic region of ProTalpha and histone H1. In the context of a physiological chromatin reconstitution reaction, the presence of ProTalpha does not affect incorporation of an amount of histone H1 sufficient to increase the nucleosome repeat length by 20 bp, but prevents association of all further H1. Consistent with this finding, a fraction of histone H1 is released when H1-containing chromatin is challenged with ProTalpha. These results imply at least two different interaction modes of H1 with chromatin, which can be distinguished by their sensitivity to ProTalpha. The properties of ProTalpha suggest a role in fine tuning the stoichiometry and/or mode of interaction of H1 with chromatin.

3T3 Cells↗
PubMed1998

Heat shock factor increases the reinitiation rate from potentiated chromatin templates.

Transcription by RNA polymerase II is highly regulated at the level of initiation and elongation. Well-documented transcription activation mechanisms, such as the recruitment of TFIID and TFIIB, control the early phases of preinitiation complex formation. The heat shock genes provide an example for transcriptional regulation at a later step: in nuclei TFIID can be detected at the TATA box prior to heat induction. Using cell-free systems for chromatin reconstitution and transcription, we have analyzed the mechanisms by which heat shock factor (HSF) increases transcription of heat shock genes in chromatin. HSF affected transcription of naked DNA templates in multiple ways: (i) by speeding up the rate of preinitiation complex formation, (ii) by increasing the number of productive templates, and (iii) by increasing the reinitiation rate. Under the more physiological conditions of potentiated chromatin templates, HSF affected only the reinitiation rate. Activator-dependent reinitiation of transcription, obviating the slow assembly of the TFIID-TFIIA complex on a promoter, may be especially crucial for genes requiring a fast response to inducers.

Animals↗
PubMed1997

The bifunctional protein DCoH modulates interactions of the homeodomain transcription factor HNF1 with nucleic acids.

The hepatocyte nuclear factor-1 (HNF1) is a homeodomain transcription factor that binds DNA as a dimer. HNF1 dimers associate with two molecules of DCoH, a bifunctional protein that also has an enzymatic function in the tetrahydrobiopterin regeneration, to form stable heterotetramers also capable of DNA binding. Employing purified, recombinant HNF1, HNF1/DCoH heterotetramers and DCoH homotetramers we investigated whether DCoH affects interactions of HNF1 with nucleic acids. Although we detected no direct binding of DCoH to DNA or RNA, DCoH stabilized HNF1/DNA complexes and promoted interactions with sub-optimal DNA target sequences such as the human alpha1-antitrypsin TATA box region. Importantly, we also observed interactions of HNF1 with RNA, but these interactions were completely abolished when HNF1 was complexed with DCoH. Interestingly, DCoH retains its enzymatic activity while complexed with HNF1. Our results document intermolecular regulation of HNF1 binding to nucleic acids by DCoH.

Animals↗
PubMed1997

Biochemical analysis of chromatin structure and function using Drosophila embryo extracts.

The biochemical analysis of chromatin structure and function is greatly facilitated by the availability of cell-free systems that assemble chromatin under physiological conditions. One such system that has shown great potential is derived from extracts of early Drosophila embryos. These embryos contain large maternal stocks of chromatin constituents, such as histones and assembly factors. Chromatin assembled in these extracts resembles native chromatin in many respects: it displays physiological nucleosome repeat lengths, it is complex, containing a wealth of nonhistone proteins as well as enzymatic activities, and it has dynamic properties that allow the interaction of DNA-binding proteins that regulate important cellular processes. Most importantly, chromatin with variant properties, e.g., with respect to the basic geometry of the nucleosomal array, histone modifications, and its content of linker histones or nonhistone proteins, can be obtained by manipulating the reconstitution conditions. The synthesis of uniform chromatin with specific characteristics should allow the analysis of the functional significance of the structural and biochemical heterogeneity observed in vivo.

Animals↗
PubMed1996

Self-organization of microtubules into bipolar spindles around artificial chromosomes in Xenopus egg extracts.

Functional nuclei and mitotic spindles are shown to assemble around DNA-coated beads incubated in Xenopus egg extracts. Bipolar spindles assemble in the absence of centrosomes and kinetochores, indicating that bipolarity is an intrinsic property of microtubules assembling around chromatin in a mitotic cytoplasm. Microtubules nucleated at dispersed sites with random polarity rearrange into two arrays of uniform polarity. Spindle-pole formation requires cytoplasmic dynein-dependent translocation of microtubules across one another. It is proposed that spindles form in the absence of centrosomes by motor-dependent sorting of microtubules according to their polarity.

Animals↗
PubMed1995

Dual regulation of the Drosophila hsp26 promoter in vitro.

Efficient heat shock induction of Drosophila hsp26 gene transcription in vivo requires binding sites for heat shock factor (HSF) and GAGA factor (GAF) close to the TATA box (proximal elements) as well as 350 bp upstream of the start site of transcription (distal elements). We have evaluated the contribution of hsp26 promoter sequences to transcriptional activity in extracts from either heat shocked or unstressed fly embryos. Efficient transcription in either extract was governed by distinct regulatory principles. Transcription in extracts from unstressed embryos relied solely on GAGA elements which efficiently counteracted repression by abundant non-specific DNA-binding proteins. Transcription in extracts from heat shocked embryos depended only a little on GAGA elements, relying mainly on functional HSEs. Constitutively active recombinant HSF or native factor in an extract from heat shocked embryos was able to truly activate transcription essentially via proximal HSEs, but not when bound to distal sites. These two modes of regulation in vitro may correspond to the two functional states of the promoter before and after heat shock in vivo.

Animals↗
PubMed1995

Chromatin remodeling by GAGA factor and heat shock factor at the hypersensitive Drosophila hsp26 promoter in vitro.

The chromatin structure at the Drosophila hsp26 promoter in vivo is characterized by two DNase I-hypersensitive (DH) sites harboring regulatory elements. Proximal and distal DH sites are separated by a positioned nucleosome. To study the contribution of transcription factors to the establishment of this specific chromatin configuration we assembled nucleosomes on the hsp26 promoter using a cell-free reconstitution system derived from fly embryos. Both DH sites were readily reconstituted from extract components. They were separated by a nucleosome which was less strictly positioned than its in vivo counterpart. The interactions of GAGA factor and heat shock factor with their binding sites in chromatin occurred in two modes. Their interaction with binding sites in the nucleosome-free regions did not require ATP. In the presence of ATP both factors interacted also with nucleosomal binding sites, causing nucleosome rearrangements and a refinement of nucleosome positions. While chromatin remodeling upon transcription factor interaction has previously been interpreted to involve nucleosome disruption, the data suggest energy-dependent nucleosome sliding as main principle of chromatin reorganization.

Animals↗
PubMed1994

Transcriptional repression by nucleosomes but not H1 in reconstituted preblastoderm Drosophila chromatin.

Chromatin reconstituted in an extract from preblastoderm Drosophila embryos represses transcription by RNA polymerase II. We have assembled regularly spaced nucleosomes on DNA attached to paramagnetic beads enabling the efficient purification of chromatin templates for transcription studies. We have used diagnostic salt extractions to establish that transcriptional repression of immobilized chromatin was largely due to nucleosome cores. When purified H1 was incorporated into chromatin, resulting in increased repeat lengths to 200-220 bp, the contribution of H1 to transcriptional repression was negligible. If more H1 was added no regularly spaced chromatin was obtained and only under these conditions was transcriptional inhibition by H1 apparent. We conclude that efficient repression of transcription by polymerase II in this system does not require the presence of histone H1.

Animals↗
PubMed1994

Nonradioactive, solid-phase DNase I footprints analyzed on an A.L.F. DNA Sequencer.

Solid-phase DNase I footprinting provides a powerful tool for analyzing the sequence-specific interactions of DNA binding proteins. Classically this type of assay requires radioactively labeled DNA molecules. Substitution of the isotope by fluorescein labeling of the DNA fragments enables the analysis of footprint patterns on a standard automated laser fluorescent (A.L.F.) DNA Sequencer. The combination of solid-phase footprinting technology and fluorescence-based nonradioactive detection of fragments has unique advantages over established footprinting technologies.

DNA-Binding Proteins↗