Biomedical subjects

O Nayler

Publications and source records attributed to O Nayler.

12 recordsLinked to original sources

PubMed2000

The ER repeat protein YT521-B localizes to a novel subnuclear compartment.

The characterization of distinct subnuclear domains suggests a dynamic nuclear framework supporting gene expression and DNA replication. Here, we show that the glutamic acid/arginine-rich domain protein YT521-B localizes to a novel subnuclear structure, the YT bodies. YT bodies are dynamic compartments, which first appear at the beginning of S-phase in the cell cycle and disperse during mitosis. Furthermore, in untreated cells of the human cell line MCF7 they were undetectable and appeared only after drug- induced differentiation. YT bodies contain transcriptionally active sites and are in close contact to other subnuclear structures such as speckles and coiled bodies. YT bodies disperse upon actinomycin D treatment, whereas other transcriptional inhibitors such as alpha-amanitin or DRB have little effect. On the basis of our experiments, we propose that YT521-B may participate in the assembly of genes into transcription centers, thereby allowing efficient regulation of gene expression.

Amino Acid Sequence↗

PubMed1999

The in vivo minigene approach to analyze tissue-specific splicing.

The exact mechanisms leading to alternative splice site selection are still poorly understood. However, recently cotransfection studies in eukaryotic cells were successfully used to decipher contributions of RNA elements (cis-factors), their interacting protein components (trans-factors) or the cell type to alternative pre-mRNA splicing. Splice factors often work in a concentration dependent manner, resulting in a gradual change of alternative splicing patterns of a minigene when the amount of a trans-acting protein is increased by cotransfections. Here, we give a detailed description of this technique that allows analysis of large gene fragments (up to 10-12 kb) under in vivo condition. Furthermore, we provide a summary of 44 genes currently investigated to demonstrate the general feasibility of this technique.

Alternative Splicing↗

PubMed1999

The interaction and colocalization of Sam68 with the splicing-associated factor YT521-B in nuclear dots is regulated by the Src family kinase p59(fyn).

Alternative pre-mRNA splicing patterns can change an extracellular stimulus, but the signaling pathways leading to these changes are still poorly characterized. Here, we describe a tyrosine-phosphorylated nuclear protein, YT521-B, and show that it interacts with the nuclear transcriptosomal component scaffold attachment factor B, and the 68-kDa Src substrate associated during mitosis, Sam68. Northern blot analysis demonstrated ubiquitous expression, but detailed RNA in situ analysis revealed cell type specificity in the brain. YT521-B protein is localized in the nucleoplasm and concentrated in 5-20 large nuclear dots. Deletion analysis demonstrated that the formation of these dots depends on the presence of the amino-terminal glutamic acid-rich domain and the carboxyl-terminal glutamic acid/arginine-rich region. We show that the latter comprises an important protein-protein interaction domain. The Src family kinase p59(fyn)-mediated tyrosine phosphorylation of Sam68 negatively regulates its association with YT521-B, and overexpression of p59(fyn) dissolves nuclear dots containing YT521-B. In vivo splicing assays demonstrated that YT521-B modulates alternative splice site selection in a concentration-dependent manner. Together, our data indicate that YT521-B and Sam68 may be part of a signal transduction pathway that influences splice site selection.

Adaptor Proteins, Signal Transducing↗

PubMed1999

ScienceLabDatabase: a computer program to organize a molecular biology laboratory.

A description of a novel laboratory management software is provided. ScienceLabDatabase (SLD) offers a useful platform to organize a molecular biology research laboratory. The program manages stocks of biological samples, including plasmids, antibodies and cell lines, laboratory protocols and addresses, and it includes an easy ordering and funds managing system. Preformed data sheet templates help to store and maintain valuable information on samples or reagents and to facilitate the transfer of accurate information between researchers. Password protection regulates access, and simple button functions allow the use of this system without prior database knowledge. SLD is based on FileMaker Pro Version 4.0, which allows easy customization and import of preexisting data from other applications. The SLD program was successfully tested in several independent research groups and proved a useful tool to efficiently organize a molecular biology laboratory.

Antibodies↗

PubMed1998

The cellular localization of the murine serine/arginine-rich protein kinase CLK2 is regulated by serine 141 autophosphorylation.

Pre-mRNA splicing is catalyzed by a multitude of proteins including serine/arginine-rich (SR) proteins, which are thought to play a crucial role in the formation of spliceosomes and in the regulation of alternative splicing. SR proteins are highly phosphorylated, and their kinases are believed to regulate the recruitment of SR proteins from nuclear storage compartments known as speckles. Recently, a family of autophosphorylating kinases termed CLK (CDC2/CDC28-like kinases) was shown to phosphorylate SR proteins and to influence alternative splicing in overexpression systems. Here we used endogenous CLK2 protein to demonstrate that it displays different biochemical characteristics compared with its overexpressed protein and that it is differentially phosphorylated in vivo. Furthermore, CLK2 changed its nuclear localization upon treatment with the kinase inhibitor 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole. We have also identified a CLK2 autophosphorylation site, which is highly conserved among all CLK proteins, and we show by site-directed mutagenesis that its phosphorylation influences the subnuclear localization of CLK2. Our data suggest that CLK2 localization and possibly activity are influenced by a balance of CLK2 autophosphorylation and the regulation by CLK2 kinases and phosphatases.

Alternative Splicing↗

PubMed1998

Human transformer-2-beta gene (SFRS10): complete nucleotide sequence, chromosomal localization, and generation of a tissue-specific isoform.

Htra2-beta is a human homologue of Drosophila transformer-2 and a member of the SR-like protein family. Here we report the isolation and characterization of the complete htra2-beta gene (HGMW-approved symbol SFRS10). The gene spans 21,232 bp and is composed of 10 exons and 9 introns. Radiation hybrid mapping localized the gene to chromosome 3q. The region upstream of the transcription initiation codon contains an Alu element and several potential transcription factor binding sites. RT-PCR and comparison with EST clones revealed five different RNA isoforms generated by alternative splicing. These isoforms encode three diverging open reading frames, and two of these, htra2-beta3 and htra2-beta4, lack the first SR domain. Htra2-beta3 is developmentally regulated and expressed predominantly in brain, liver testis, and weakly in kidney. Furthermore, the domain structure of htra2-beta3 resembles a variant found in the Drosophila male germline, indicating a remarkable conservation of alternative transformer-2 variants. Finally, we show that htra2-beta3 is expressed in the nucleus and interacts with a subset of SR proteins in a yeast two-hybrid system and in vivo.

Alternative Splicing↗

PubMed1998

SAF-B protein couples transcription and pre-mRNA splicing to SAR/MAR elements.

Interphase chromatin is arranged into topologically separated domains comprising gene expression and replication units through genomic sequence elements, so-called MAR or SAR regions (for matrix- or scaffold-associating regions). S/MAR regions are located near the boundaries of actively transcribed genes and were shown to influence their activity. We show that scaffold attachment factor B (SAF-B), which specifically binds to S/MAR regions, interacts with RNA polymerase II (RNA pol II) and a subset of serine-/arginine-rich RNA processing factors (SR proteins). SAF-B localized to the nucleus in a speckled pattern that coincided with the distribution of the SR protein SC35. Furthermore, we show that overexpressed SAF-B induced an increase of the 10S splice product using an E1A reporter gene and repressed the activity of an S/MAR flanked CAT reporter gene construct in vivo . This indicates an association of SAF-B with SR proteins and components of the transcription machinery. Our results describe the coupling of a chromatin organizing S/MAR element with transcription and pre-mRNA processing components and we propose that SAF-B serves as a molecular base to assemble a 'transcriptosome complex' in the vicinity of actively transcribed genes.

3T3 Cells↗

PubMed1997

Characterization and comparison of four serine- and arginine-rich (SR) protein kinases.

Phosphorylated serine- and arginine-rich (SR) proteins are components of the spliceosomal complex, and have been implicated in the control of alternative splicing. Kinases that regulate the phosphorylation and possibly the intranuclear distribution of SR proteins may therefore contribute to changes in choice of splice site. We have cloned three mouse cDNAs with high sequence identity to the family of LAMMER kinases (i.e. kinases carrying the conserved signature EHLAMMERILG in the catalytic domain). A comparison of their amino acid sequences revealed two related subfamilies with high evolutionary conservation. We have compared the expression patterns of these proteins in mouse tissues and transformed cell lines with that of a previously cloned family member (mCLK1/STY), and detected various transcripts for each gene. This underlines previous findings of alternative splicing of mclk1/STY. Our results suggest that the proportions of products for each gene are regulated independently. We further demonstrate that all variants encode autophosphorylating proteins that can phosphorylate several biochemically purified SR proteins in vitro, leading to hyperphosphorylation of at least one SR protein in vivo. The observed tissue distributions and substrate specificities suggest that these kinases may all be constituents of a network of regulatory mechanisms that enable SR proteins to control RNA splicing.

Amino Acid Sequence↗

PubMed1995

The proximal pathway of metabolism of the chlorinated signal molecule differentiation-inducing factor-1 (DIF-1) in the cellular slime mould Dictyostelium.

Stalk cell differentiation during development of the slime mould Dictyostelium is induced by a chlorinated alkyl phenone called differentiation-inducing factor-1 (DIF-1). Inactivation of DIF-1 is likely to be a key element in the DIF-1 signalling system, and we have shown previously that this is accomplished by a dedicated metabolic pathway involving up to 12 unidentified metabolites. We report here the structure of the first four metabolites produced from DIF-1, as deduced by m.s., n.m.r. and chemical synthesis. The structures of these compounds show that the first step in metabolism is a dechlorination of the phenolic ring, producing DIF metabolite 1 (DM1). DM1 is identical with the previously known minor DIF activity, DIF-3. DIF-3 is then metabolized by three successive oxidations of its aliphatic side chain: a hydroxylation at omega-2 to produce DM2, oxidation of the hydroxy group to a ketone group to produce DM3 and a further hydroxylation at omega-1 to produce DM4, a hydroxyketone of DIF-3. We have investigated the enzymology of DIF-1 metabolism. It is already known that the first step, to produce DIF-3, is catalysed by a novel dechlorinase. The enzyme activity responsible for the first side-chain oxidation (DIF-3 hydroxylase) was detected by incubating [3H]DIF-3 with cell-free extracts and resolving the reaction products by t.l.c. DIF-3 hydroxylase has many of the properties of a cytochrome P-450. It is membrane-bound and uses NADPH as co-substrate. It is also inhibited by CO, the classic cytochrome P-450 inhibitor, and by several other cytochrome P-450 inhibitors, as well as by diphenyliodonium chloride, an inhibitor of cytochrome P-450 reductase. DIF-3 hydroxylase is highly specific for DIF-3: other closely related compounds do not compete for the activity at 100-fold molar excess, with the exception of the DIF-3 analogue lacking the chlorine atom. The Km for DIF-3 of 47 nM is consistent with this enzyme being responsible for DIF-3 metabolism in vivo. The two further oxidations necessary to produce DM4 are also performed in vitro by similar enzyme activities. One of the inhibitors of DIF-3 hydroxylase, ancymidol (IC50 67 nM) is likely to be particularly suitable for probing the function of DIF metabolism during development.

Animals↗

PubMed1993

A localized differentiation-inducing-factor sink in the front of the Dictyostelium slug.

Differentiation-inducing factor 1 [DIF-1; 1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)-hexan-1-one] induces stalk cell differentiation during Dictyostelium development. It is present as a gradient in the multicellular slug, its lowest concentration being in the anterior. Here we demonstrate the existence of a localized sink for DIF-1, also in the anterior of the slug, which could be responsible for generating the DIF-1 gradient. DIF-1 is metabolized extensively by developing cells, initially by a mono-dechlorination. We used an enzyme assay for DIF-1 dechlorinase to examine its distribution in the slug. DIF-1 dechlorinase activity is 30-fold higher in prestalk cells (largely anterior) compared with prespore cells (posterior) when these are separated from each other on Percoll density gradients. Dissection experiments showed that DIF-1 dechlorinase is 25-fold enriched in the anterior 13% of the slug compared with the rest. These experiments also showed that DIF-1 dechlorinase is more anterior-enriched than the standard prestalk markers, the ecmA and ecmB mRNAs. When cut from a slug, both prestalk and prespore fragments regulate to restore the missing cell type. Prespore fragments rapidly regain (by 30 min) a DIF-1 sink in their anteriors, and prestalk fragments restore a posterior zone with low DIF-1 dechlorinase by 4 hr after cutting. The reappearance of the DIF-1 sink in the anterior of prespore fragments is accomplished without detectable cell sorting and may, therefore, be in response to positional signals. Finally, a localized sink may provide a general way of producing a gradient of a signal substance in a developing embryo.

Animals↗

PubMed1992

Differentiation-inducing-factor dechlorinase, a novel cytosolic dechlorinating enzyme from Dictyostelium discoideum.

Differentiation-inducing factor 1 (DIF-1) is a dichlorinated alkyl phenone (1-[(3,5-dichloro-2,6-dihydroxy-4-methoxy)phenyl]hexan-1-one) from Dictyostelium discoideum, that induces amoebae to differentiate into stalk cells. It was shown previously that DIF-1 is rapidly metabolized into a series of more polar compounds by living cells [Traynor, D. & Kay, R.R. (1991) J. Biol. Chem. 266, 5291-5297]. The first step in DIF-1 metabolism is the formation of DIF metabolite 1 (now known to be DIF-3) by a monodechlorination. We report here the discovery of the enzyme activity catalyzing this dechlorination. A very sensitive enzyme assay was developed, using [3H]DIF-1 and a TLC system to separate DIF-1 from the product, DIF-3. DIF-1 3(5)-dechlorinase is present in the high-speed supernatant of cell lysates, and uses glutathione, at physiological concentrations, as cofactor. Kinetic measurements indicate a Km for DIF-1 of about 70 nM. The enzyme activity is inhibited by DIF-2 (the pentan-1-one analogue of DIF-1), with a median inhibitor concentration (IC50) of 1 microM, and DIF-3 (IC50 = 5 microM), which presumably act as substrates, but other compounds structurally related to DIF-1 were much less effective. Aurothioglucose, an inhibitor of selenocysteine enzymes, inhibited DIF-1 3(5)-dechlorinase with IC50 = 100 nM. DIF-1 3(5)-dechlorinase activity is developmentally regulated. It is essentially absent from growing cells and increases at the end of aggregation to reach a first peak of activity at the first finger stage, with a further rise at culmination.

Animals↗

PubMed1992

DIF-1 induces its own breakdown in Dictyostelium.

DIF-1 is a novel chlorinated alkyl phenone which induces differentiation of prestalk cells in Dictyostelium discoideum. It is broken down and inactivated by a cytoplasmic enzyme, DIF-1 3(5)-dechlorinase (hereafter referred to as DIF-1 dechlorinase), which is found only in prestalk cells. We show that DIF-1 dechlorinase levels are induced at least 50-fold when cells are treated with DIF-1. This response is rapid--enzyme activity doubles within 15 min and is fully induced within an hour--and occurs early in development, before other prestalk markers can be induced by DIF-1. Maximum inducibility is seen towards the end of aggregation, when DIF-1 dechlorinase is barely detectable in uninduced cells. The dose-dependence reveals a threshold concentration of DIF-1 (15 nM) below which almost no response is seen. Cyclic AMP, which is the chemoattractant during aggregation and plays a key role in later development, suppresses the induction of DIF-1 dechlorinase by DIF-1. We conclude that induction of DIF-1 dechlorinase is one of the first steps on the developmental pathway which leads to prestalk cell differentiation, and suggest that the resulting negative feedback on DIF-1 levels is an important part of the mechanism by which cells decide whether to become prestalk or prespore cells.

Animals↗