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D A Lafontaine

Publications and source records attributed to D A Lafontaine.

17 recordsLinked to original sources
PubMed2002

The structure and active site of the Varkud satellite ribozyme.

The Varkud satellite ribozyme is the largest of the small nucleolytic ribozymes, and the only one for which there is no crystal structure. It can be divided into a trans -acting ribozyme, consisting of five helices organized by two three-way helical junctions, and a stem-loop substrate with which it interacts, primarily by tertiary interactions. We have determined the global fold of the ribozyme, and the manner by which it interacts with the substrate. The substrate interacts with a cleft formed between helices II and VI (organized by the lower helical junction), where it contacts the A730 loop, the probable active site of the ribozyme. Within this loop, there is a critical adenine base (A756) that is a candidate for direct nucleobase participation in the cleavage reaction.

Base Sequence↗
PubMed2001

The A730 loop is an important component of the active site of the VS ribozyme.

The core of the VS ribozyme comprises five helices, that act either in cis or in trans on a stem-loop substrate to catalyse site-specific cleavage. The structure of the 2-3-6 helical junction indicates that a cleft is formed between helices II and VI that is likely to serve as a receptor for the substrate. Detailed analysis of sequence variants suggests that the base bulges of helices II and VI play an architectural role. By contrast, the identity of the nucleotides in the A730 loop is very important for ribozyme activity. The base of A756 is particularly vital, and substitution by any other nucleotide or ablation of the base leads to a major reduction in cleavage rate. However, variants of A756 bind substrate efficiently, and are not defective in global folding. These results suggest that the A730 loop is an important component of the active site of the ribozyme, and that A756 could play a key role in catalysis.

Adenine↗
PubMed2001

Structure, folding and activity of the VS ribozyme: importance of the 2-3-6 helical junction.

The VS nucleolytic ribozyme has a core comprising five helices organized by two three-way junctions. The ribozyme can act in trans on a hairpin-loop substrate, with which it interacts via tertiary contacts. We have determined that one of the junctions (2-3-6) undergoes two-stage ion-dependent folding into a stable conformation, and have determined the global structure of the folded junction using long-range distance restraints derived from fluorescence resonance energy transfer. A number of sequence variants in the junction are severely impaired in ribozyme cleavage, and there is good correlation between changes in activity and alteration in the folding of junction 2-3-6. These studies point to a special importance of G and A nucleotides immediately adjacent to helix II, and comparison with a similar junction of known structure indicates that this could adopt a guanine-wedge structure. We propose that the 2-3-6 junction organizes important aspects of the structure of the ribozyme to facilitate productive association with the substrate, and suggest that this results in an interaction between the substrate and the A730 loop to create the active complex.

Adenine↗
PubMed2000

Nucleotides -1 to -4 of hepatitis delta ribozyme substrate increase the specificity of ribozyme cleavage.

In the past, the use of delta ribozyme as a therapeutic tool was limited because substrate specificity was thought to be determined by only 8 nucleotides. Recently, we have accumulated evidence suggesting that the substrate sequence upstream of the cleavage site, which is not involved in the binding with the delta ribozyme, appears to be essential in the selection of an appropriate cleavage site. To understand the role of this region in efficient cleavage, we synthesized a collection of small substrates that possessed single and multiple mutations in positions -1 to -4 and determined the kinetic parameters of their cleavage using a model antigenomic delta ribozyme. Some substrates were found to be uncleavage, whereas others showed >60-fold difference in relative specificity between the least and most efficiently cleaved substrates. The base at each position from -1 to -4 contributes differently to the ability of a substrate to be cleaved. An optimal sequence for positions -1 to -4 was determined to be -1HRHY(-4) (H = U, C, or A). These results shed light on new features that contribute to the substrate requirement of delta ribozyme cleavage and should increase interest in the use of this unique ribozyme.

Base Sequence↗
PubMed1999

Presence of a coordinated metal ion in a trans-acting antigenomic delta ribozyme.

We have investigated the cleavage induced by metal ions in an antigenomic form of a trans-acting delta ribozyme. A specific Mg(2+)-induced cleavage at position G(52)at the bottom of the P2 stem was observed to occur solely within catalytically active ribozyme-substrate complexes (i.e. those that performed the essential conformational transition step). Only the divalent cations which support catalytic activity permitted the detection of specific induced cleavages in this region. Using various mutant ribozymes and substrates, we demonstrated a correlation between enzymatic activity and the Mg(2+)-induced cleavage pattern. We show that the efficiency of the coordination of the magnesium to its binding site is related to the nature of the base pair in the middle of the P1 stem (i.e. Rz(23)-S(8)). Together with additional evidence from nuclease probing experiments that indicates the occurrence of a structural rearrangement involving the bottom of the P2 stem upon formation of the P1 helix, these results show that an intimate relationship exists between the folding and the catalytic activity of the delta ribozyme.

Base Sequence↗
PubMed1999

Mutational analysis of the antigenomic trans-acting delta ribozyme: the alterations of the middle nucleotides located on the P1 stem.

Our previous report on delta ribozyme cleavage using a trans -acting antigenomic delta ribozyme and a collection of short substrates showed that the middle nucleotides of the P1 stem, the substrate binding site, are essential for the cleavage activity. Here we have further investigated the effect of alterations in the P1 stem on the kinetic and thermodynamic parameters of delta ribozyme cleavage using various ribozyme variants carrying single base mutations at putative positions reported. The kinetic and thermodynamic values obtained in mutational studies of the two middle nucleotides of the P1 stem suggest that the binding and active sites of the delta ribozyme are uniquely formed. Firstly, the substrate and the ribozyme are engaged in the formation of a helix, known as the P1 stem, which may contain a weak hydrogen bond(s) or a bulge. Secondly, a tertiary interaction involving the base moieties in the middle of the P1 stem likely plays a role in defining the chemical environment. As a con-sequence, the active site might form simultaneously or subsequently to the binding site during later steps of the pathway.

Binding Sites↗
PubMed1999

The viroid and viroid-like RNA database.

This is an online database to facilitate research on viroid, viroid-like RNAs and human hepatitis delta virus (vHDV) by presenting a large number of sequences and related data in a comprehensive and user-friendly format (e.g. position of their self-catalytic domains, open reading frame of the vHDV, prediction of the most stable secondary structures, etc.). Most of these RNA species share a common proposed replication pattern known as a DNA-independent rolling circle mechanism. Together, these species form the 'brotherhood' of the smallest known auto-replicable RNAs. This online database is available on the World Wide Web at http://www.callisto.si.usherb.ca/jpperra

Databases, Factual↗
PubMed1998

The viroid and viroid-like RNA database.

The viroid and viroid-like RNA database is a compilation of all natural sequences published in journals or available from the GenBank and EMBL nucleotide sequence libraries. Several information regarding these RNA species such as the position of their self-catalytic domains and the open reading frame of the human hepatitits delta virus are provided. The database also includes a determination of the likely ancestral sequence of most species and a prediction of the most stable secondary structures of these sequences. This online database is available on the World Wide Web (http://www.callisto.si.usherb.ca/[symbol: see text]jpperra ). It should provide an excellent reference point for further phylogenetic and structure-function studies of these RNA species.

Computer Communication Networks↗
PubMed1998

Identification of a Crohn's disease specific transcript with potential as a diagnostic marker.

BACKGROUND: A long time goal of the medical research community has been the identification of a reliable and valid marker for Crohn's disease. AIM: To identify differences in the genetic expression patterns of healthy and diseased tissues. METHOD: The RNA arbitrarily primed polymerase chain reaction (RAP-PCR) procedure was modified to improve its potential to identify clinical markers in heterogeneous RNA populations. RESULTS: With this procedure, a 1065 bp PCR product associated with the inflammation that occurs in Crohn's disease was identified, cloned and sequenced. Northern blot hybridisations showed that this novel sequence originates from a unique RNA species of 3.1 kb. Dot blot hybridisations clearly showed that this RNA species was specific to Crohn's disease. Moreover, its abundance seemed to correlate with the severity of inflammation. Finally, this RNA species was also detected in macroscopically normal areas from Crohn's disease specimens, suggesting that it appears either early during the disease or at least before severe manifestations. CONCLUSION: This finding of a 3.1 kb RNA species permits the discrimination of Crohn's disease manifestations. Although further clinical work is required, this transcript appears to have definite potential as a diagnostic marker.

Base Sequence↗
PubMed1992

Characterization of an alternative promoter in the human growth hormone gene.

Transcription of the human growth hormone (hGH) gene depends on cis-acting elements contained within 300 base pairs of its 5'-flanking sequence. An earlier in vitro study of the transcriptional activity of this 5'-flanking region suggested that transcription can start upstream from position +1. We have investigated this phenomenon by cell-free transcription and transient transfection of chimeric constructs in cultured pituitary cells and in HeLa cells and by analysis of RNA from human pituitary glands and HeLa cells. Transcription initiation sites were identified at positions -54 and -197 by cell-free transcription assays and by RNAse mapping of human pituitary RNA. In transfection assays, the hGH gene 5'-flanking sequence upstream from position -197 displayed transcriptional activity, which critically depended on the upstream stimulatory factor-binding site located between positions -253 and -266. Transcripts initiated upstream from position +1 were detected in human pituitary RNA by polymerase chain reaction amplification and Northern blotting. These transcripts were longer than the mRNA encoding hGH. They might control initiation at position +1 or code for a novel peptide.

Base Sequence↗
PubMed1990

Nuclear factor-I and activator protein-2 bind in a mutually exclusive way to overlapping promoter sequences and trans-activate the human growth hormone gene.

Transcription of the human growth hormone (hGH) gene and its regulation are controlled by trans-acting factors that bind to hGH gene promoter sequences. Several DNase I footprints have been described within 500 bp of this promoter, one of which (-289 to -267) has not yet been ascribed to a defined factor. By DNase I footprinting, gel mobility shift, and methylation interference assays with extracts from HeLa cells and GH-producing pituitary tumor (GC) cells, we show that this factor belongs to the NF-I family. When NF-I was competed out of the cell extracts, the trans-acting factor AP-2 bound to the same site as NF-I. AP-2 was present not only in HeLa cells, but also in GC cells albeit at a much lower concentration. Consistent with the mutually exclusive binding of NF-I and AP-2, their methylation interference patterns included four guanine residues that were crucial for binding of both NF-I and AP-2. Cell-free transcription from the hGH gene promoter showed that these two factors can transactivate this gene.

Base Sequence↗
PubMed1990

Sp1 can displace GHF-1 from its distal binding site and stimulate transcription from the growth hormone gene promoter.

DNase I footprinting experiments showed that binding activities of Sp1 and of GHF-1 to its distal site on the human growth hormone gene promoter are mutually exclusive. The kinetics of GHF-1 binding were indicative of positive cooperativity. The Sp1 site did not affect promoter activity in cell-free transcription. Still, Sp1 could compensate partially for the decreased stimulation of transcription seen at low GHF-1 concentrations.

Base Sequence↗
PubMed1989

Evidence that the upstream stimulatory factor and the Sp1 transcription factor bind in vitro to the promoter of the human-growth-hormone gene.

Expression of the human-growth-hormone gene is restricted to pituitary somatotrophs. Two protein-DNA complexes that are specific to the pituitary, and two that are not, had been demonstrated in vitro on the promoter of this gene. The two pituitary-specific footprints had been ascribed to a single protein called growth hormone factor 1. We have now characterized the factors responsible for the two other footprints by means of deoxyribonuclease-I protection and gel-retardation experiments. The first footprint, located between -257 and -290 relative to the transcription initiation site, involves at least two factors present in pituitary cells. One of these factors binds between nucleotides -257 and -267, and is indistinguishable from the upstream stimulatory factor, also called major late transcription factor or upstream element factor, initially described in HeLa cells. Earlier work by others had shown that the activator protein 2 purified from HeLa cells can bind to nucleotides -263 and -290. Our experiments suggest that a factor different from activator protein 2 is involved in the protection of this region against deoxyribonuclease I. The second footprint, located between nucleotides -116 and -140, involves only one factor. This factor, present in pituitary cells, recognizes a GC box and is indistinguishable from transcription factor Sp1, previously described in HeLa cells. The human-growth-hormone gene is therefore a candidate for regulation by these factors in vivo.

Animals↗
PubMed1989

Pituitary-specific factor binding to the human prolactin, growth hormone, and placental lactogen genes.

The human genes coding for growth hormone (GH), chorionic somatomammotropin (placental lactogen, CS), and prolactin (Prl) are related evolutionarily but are expressed in phenotypically distinct cell types despite their nucleotide sequence homology. We show here that the promoters of the human Prl and CS genes contain cis-acting sequences that confer pituitary-specific expression in a cell-free transcription assay. Similar data are obtained with the human GH gene, consistent with earlier work by others. Footprinting analysis shows that neighboring sequences in each of these three promoters are protected from deoxyribonuclease I digestion by rat pituitary cell extracts. Footprinting competition experiments and gel retardation assays with synthetic oligonucleotides suggest that a single factor is responsible for the pituitary-specific footprints seen on the human Prl, CS, and GH genes. They also suggest that this factor is identical or closely related to the trans-acting factor GHF-1/Pit-1. Similarities with and differences from the rat GH and Prl genes are discussed.

Animals↗
PubMed1987

Glucocorticoid receptors bound to the antagonist RU486 are not downregulated despite their capacity to interact in vitro with defined gene regions.

Modulation of gene expression by glucocorticoids involves interaction of these hormones with an intracellular receptor followed by 'transformation' of the hormone-receptor complex into a nuclear binding form. The molecular basis for the antiglucocorticoid action of high-affinity steroid analogues such as RU486 remains controversial. The effects of dexamethasone and RU486 on in vitro and in vivo properties of the receptor were compared using human lymphoblastoid IM-9 cells. In these cells, RU486 fully antagonized the glucocorticoid-specific induction of 5'-nucleotidase activity by dexamethasone. In vitro, however, RU486-bound receptor could be transformed and shown to interact specifically with cloned DNA fragments containing glucocorticoid response elements. These fragments included one from the mouse mammary tumour virus and two from the human growth hormone gene. In vivo, RU486-bound receptor did not behave like dexamethasone-bound receptor. While receptor downregulation, a property of the transformed receptor, was achieved by dexamethasone, this did not occur with RU486. Likewise, RU486 did not affect receptor half-life under conditions when this was shortened by dexamethasone. These seemingly contradictory results can be reconciled by proposing that receptor transformation by agonists involves dissociation of the receptor oligomer to reveal a DNA-binding site that pre-exists on this protein. Although cell-free receptor dissociation and therefore DNA binding can occur even when the receptor is bound to RU486, this steroid maintains receptors in the untransformed state in the intact cell and therefore behaves a glucocorticoid antagonist in vivo.

5'-Nucleotidase↗
PubMed1987

Approach to the molecular mechanisms of the modulation of growth hormone gene expression by glucocorticoid and thyroid hormones.

Glucocorticoid and thyroid hormones modulate the expression of the growth hormone gene. To investigate this control mechanism, we have determined whether this gene contains sites that bind the human glucocorticoid and thyroid hormone receptors in vitro. To do so, we have designed a novel assay for studying binding of the purified glucocorticoid receptor to cloned fragments of the human growth hormone (hGH) gene, and have adapted a DNA-competition assay for the thyroid receptor in nuclear cell extracts. Two glucocorticoid receptor binding regions were found in the hGH gene, one of high affinity in a fragment of the gene containing the first intron, and one of low affinity located within a 290 bp-fragment of 5'-flanking DNA. In contrast, the thyroid receptor bound with high affinity to the 5'-flanking fragment. Homologous binding regions for the two types of receptor were found in the human placental lactogen (chronic somatomammotropin) gene. DNA binding of the two receptor types appeared to depend on the presence of the hormone, yet antagonist-bound glucocorticoid receptor was still capable of interacting specifically with DNA. There was no evidence for synergism or antagonism of the two receptor types in binding to their respective sites on the hGH gene. The data also make it unlikely that the thyroid receptor negatively controls gene transcription and that the stimulatory effect of thyroid hormone results from a derepression mechanism.

Animals↗