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S Stamm

Publications and source records attributed to S Stamm.

At least 37 records · Page 2Linked to original sources
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

Activity-dependent regulation of alternative splicing patterns in the rat brain.

Alternative splicing plays an important role in the expression of genetic information. Among the best understood alternative splicing factors are transformer and transformer-2, which regulate sexual differentiation in Drosophila. Like the Drosophila genes, the recently identified mammalian homologues are subject to alternative splicing. Using an antibody directed against the major human transformer-2 beta isoform, we show that it has a widespread expression in the rat brain. Pilocarpine-induced neuronal activity changes the alternative splicing pattern of the human transformer-2-beta gene in the brain. After neuronal stimulation, a variant bearing high similarity to a male-specific Drosophila tra-2179 isoform is switched off in the hippocampus and is detectable in the cortex. In addition, the ratio of another short RNA isoform (htra2-beta2) to htra2-beta1 is changed. Htra2-beta2 is not translated into protein, and probably helps to regulate the relative amounts of htra2-beta1 to beta3. We also observe activity-dependent changes in alternative splicing of the clathrin light chain B, c-src and NMDAR1 genes, indicating that the coordinated change of alternative splicing patterns might contribute to molecular plasticity in the brain.

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↗
PubMed1997

Molecular cloning of a novel alternatively spliced nuclear protein.

Using the yeast two hybrid system, we isolated a rat cDNA (E3-3) coding for a new protein with no homology to any other protein in the database. E3-3 is ubiquitously expressed. Variants that most likely arise through alternative splicing encode truncated forms of the protein. Testis is the only tissue that predominantly expresses the longest protein variant. When this variant is tagged with enhanced green fluorescent protein, the protein is located in the nucleus.

Alternative Splicing↗
PubMed1997

Molecular cloning of htra2-beta-1 and htra2-beta-2, two human homologs of tra-2 generated by alternative splicing.

A yeast two-hybrid screen was performed to find new factors involved in pre-mRNA splicing. Using SC35 as a bait, we isolated a human cDNA bearing high homology to the Drosophila transformer-2 (TRA-2) protein. This cDNA was named htra2-beta1. htra2-beta1 is a nuclear protein that colocalizes with SC35 in a speckled pattern. It interacts with several SR proteins tested in yeast. A second form named htra2-beta2 is generated by alternative splicing. This isoform gives rise to a truncated protein without an SR domain. Both isoforms are evenly distributed throughout adult rat tissue. The ratio of these two isoforms changes after stimulation of primary human T-cell and primary rat spleen cell cultures, indicating that alternative splicing is involved in regulation of htra2-beta activity.

Alternative Splicing↗
PubMed1995

Laser surgery for retinopathy of prematurity.

Retinopathy of prematurity (ROP) is a common eye disorder occurring in preterm infants. Formally known as retrolental fibroplasia, ROP is defined as a vasoproliferative disorder that occurs in the developing retina of premature infants' eyes. Until recently, cryotherapy was the recommended treatment for infants diagnosed with advancing ROP. Now laser surgery is being used as an alternative treatment. This article reviews the pathophysiology, etiology, and classification of ROP, considers the pros and cons of laser surgery versus cryotherapy, and outlines the experience with laser surgery in our NICU.

Humans↗
PubMed1994

Regulation of alternative splicing in vivo by overexpression of antagonistic splicing factors.

The opposing effects of SF2/ASF and heterogeneous nuclear ribonucleoprotein (hnRNP) A1 influence alternative splicing in vitro. SF2/ASF or hnRNP A1 complementary DNAs were transiently overexpressed in HeLa cells, and the effect on alternative splicing of several cotransfected reporter genes was measured. Increased expression of SF2/ASF activated proximal 5' splice sites, promoted inclusion of a neuron-specific exon, and prevented abnormal exon skipping. Increased expression of hnRNP A1 activated distal 5' splice sites. Therefore, variations in the intracellular levels of antagonistic splicing factors influence different modes of alternative splicing in vivo and may be a natural mechanism for tissue-specific or developmental regulation of gene expression.

Adenovirus E1A Proteins↗
PubMed1994

A sequence compilation and comparison of exons that are alternatively spliced in neurons.

Alternative splicing is an important regulatory mechanism to create protein diversity. In order to elucidate possible regulatory elements common to neuron specific exons, we created and statistically analysed a database of exons that are alternatively spliced in neurons. The splice site comparison of alternatively and constitutively spliced exons reveals that some, but not all alternatively spliced exons have splice sites deviating from the consensus sequence, implying diverse patterns of regulation. The deviation from the consensus is most evident at the -3 position of the 3' splice site and the +4 and -3 position of the 5' splice site. The nucleotide composition of alternatively and constitutively spliced exons is different, with alternatively spliced exons being more AU rich. We performed overlapping k-tuple analysis to identify common motifs. We found that alternatively and constitutively spliced exons differ in the frequency of several trinucleotides that cannot be explained by the amino acid composition and may be important for splicing regulation.

Alternative Splicing↗
PubMed1993

Brain-specific tropomyosins TMBr-1 and TMBr-3 have distinct patterns of expression during development and in adult brain.

In this study we report on the developmental and regional expression of two brain-specific isoforms of tropomyosin, TMBr-1 and TMBr-3, that are generated from the rat alpha-tropomyosin gene via the use of alternative promoters and alternative RNA splicing. Western blot analysis using an exon-specific peptide polyclonal antibody revealed that the two isoforms are differentially expressed in development with TMBr-3 appearing in the embryonic brain at 16 days of gestation, followed by the expression of TMBr-1 at 20 days after birth. TMBr-3 was detected in all brain regions examined, whereas TMBr-1 was detected predominantly in brain areas that derived from the prosencephalon. Immunocytochemical studies on mixed primary cultures made from rat embryonic midbrain indicate that expression of the brain-specific epitope is restricted to neurons. The developmental pattern and neuronal localization of these forms of tropomyosin suggest that these isoforms have a specialized role in the development and plasticity of the nervous system.

Aging↗
PubMed1993

The buccalin-related neuropeptides: isolation and characterization of an Aplysia cDNA clone encoding a family of peptide cotransmitters.

The buccalin-related peptides, buccalin A and buccalin B, are members of a family of cotransmitters that modulate neuromuscular transmission in Aplysia. In this study, a third buccalin-related peptide, buccalin C, was purified from neuronal elements in the accessory radula closer, a muscle involved in the animal's feeding behavior. Oligonucleotide probes based upon the amino acid sequence of buccalin C were used to isolate cDNA clones that encode a buccalin precursor polypeptide. The buccalin precursor contains 19 distinct buccalin-related peptides, several of which are present in multiple copies. The buccalin gene appears to be present in a single copy, with one allele containing a small insert. Expression of this gene occurs in a tissue-specific manner and mRNA transcripts are abundant within neurons in the Aplysia CNS. This large family of neuropeptides may exert extraordinarily complex modulatory actions at synapses where they serve as cotransmitters.

Amino Acid Sequence↗
PubMed1993

The myomodulin-related neuropeptides: characterization of a gene encoding a family of peptide cotransmitters in Aplysia.

The myomodulin-related peptides comprise a family of cotransmitters that modulate neuromuscular signaling in the feeding system of Aplysia. In this study, cDNA clones encoding a myomodulin precursor polypeptide were isolated and characterized. This precursor contains seven different myomodulin-related peptides, one of which, myomodulin A, is present in 10 contiguous copies. The sequence of a myomodulin genomic clone indicates that all of these myomodulin-related peptides are encoded on a single exon. The myomodulin gene is expressed in a tissue-specific manner and myomodulin mRNA is localized to specific neurons in the Aplysia CNS. The presence of multiple related neuropeptides can greatly increase the range and precision of signaling at synapses where they act as modulator cotransmitters.

Amino Acid Sequence↗
PubMed1992

Clathrin light chain B: gene structure and neuron-specific splicing.

The clathrin light chains are components of clathrin coated vesicles, structural constituents involved in endocytosis and membrane recycling. The clathrin light chain B (LCB) gene encodes two isoforms, termed LCB2 and LCB3, via an alternative RNA splicing mechanism. We have determined the structure of the rat clathrin light chain B gene. The gene consists of six exons that extend over 11.9 kb. The first four exons and the last exon are common to the LCB2 and LCB3 isoforms. The fifth exon, termed EN, is included in the mRNA in brain, giving rise to the brain specific form LCB2 but is excluded in other tissues, generating the LCB3 isoform. Primary rat neuronal cell cultures express predominantly the brain specific LCB2 isoform, whereas primary rat cultures of glia express only the LCB3 isoform, suggesting that expression of the brain-specific LCB2 form is limited to neurons. Further evidence for neuronal localization of the LCB2 form is provided using a teratocarcinoma cell line, P19, which can be induced by retinoic acid to express a neuronal phenotype, concomitant with the induction of the LCB2 form. In order to determine the sequences involved in alternative splice site selection, we constructed a minigene containing the alternative spliced exon EN and its flanking intron and exon sequences. This minigene reflects the splicing pattern of the endogenous gene upon transfection in HeLa cell and primary neuronal cell cultures, indicating that this region of the LCB gene contains all the necessary information for neuron-specific splicing.

Alternative Splicing↗