Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Mutually exclusive mutation”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 289 records · Page 16Linked to original sources
PubMed2001

A purine-rich intronic element enhances alternative splicing of thyroid hormone receptor mRNA.

The mammalian thyroid hormone receptor gene c-erbAalpha gives rise to two mRNAs that code for distinct isoforms, TRalpha1 and TRalpha2, with antagonistic functions. Alternative processing of these mRNAs involves the mutually exclusive use of a TRalpha1-specific polyadenylation site or TRalpha2-specific 5' splice site. A previous investigation of TRalpha minigene expression defined a critical role for the TRalpha2 5' splice site in directing alternative processing. Mutational analysis reported here shows that purine residues within a highly conserved intronic element, SEa2, enhance splicing of TRalpha2 in vitro as well as in vivo. Although SEalpha2 is located within the intron of TRalpha2 mRNA, it activates splicing of a heterologous dsx pre-mRNA when located in the downstream exon. Competition with wild-type and mutant RNAs indicates that SEalpha2 functions by binding trans-acting factors in HeLa nuclear extract. Protein-RNA crosslinking identifies several proteins, including SF2/ASF and hnRNP H, that bind specifically to SEalpha2. SEalpha2 also includes an element resembling a 5' splice site consensus sequence that is critical for splicing enhancer activity. Mutations within this pseudo-5' splice site sequence have a dramatic effect on splicing and protein binding. Thus SEa2 and its associated factors are required for splicing of TRalpha2 pre-mRNA.

Alternative Splicing↗
PubMed2006

Analysis of the ribosomal protein S19 interactome.

Ribosomal protein S19 (RPS19) is a 16-kDa protein found mainly as a component of the ribosomal 40 S subunit. Its mutations are responsible for Diamond Blackfan anemia, a congenital disease characterized by defective erythroid progenitor maturation. Dysregulation of RPS19 has therefore been implicated in this defective erythropoiesis, although the link between them is still unclear. Two not mutually exclusive hypotheses have been proposed: altered protein synthesis and loss of unknown functions not directly connected with the structural role of RPS19 in the ribosome. A role in rRNA processing has been surmised for the yeast ortholog, whereas the extracellular RPS19 dimer has a monocyte chemotactic activity. Three proteins are known to interact with RPS19: FGF2, complement component 5 receptor 1, and a nucleolar protein called RPS19-binding protein. We have used a yeast two-hybrid approach to identify a fourth protein: the serine-threonine kinase PIM1. The present study describes our use of proteomics strategies to look for proteins interacting with RPS19 to determine its functions. Proteins were isolated by affinity purification with a GST-RPS19 recombinant protein and identified using LCMS/MS analysis coupled to bioinformatics tools. We identified 159 proteins from the following Gene Ontology categories: NTPases (ATPases and GTPases; five proteins), hydrolases/helicases (19 proteins), isomerases (two proteins), kinases (three proteins), splicing factors (five proteins), structural constituents of ribosome (29 proteins), transcription factors (11 proteins), transferases (five proteins), transporters (nine proteins), DNA/RNA-binding protein species (53 proteins), other (one dehydrogenase protein, one ligase protein, one peptidase protein, one receptor protein, and one translation elongation factor), and 13 proteins of still unknown function. Proteomics results were validated by affinity purification and Western blotting. These interactions were further confirmed by co-immunoprecipitation using a monoclonal RPS19 antibody. Many interactors are nucleolar proteins and thus are expected to take part in the RPS19 interactome; however, some proteins suggest additional functional roles for RPS19.

Glutathione Transferase↗
PubMed1995

Association of p120, a tyrosine kinase substrate, with E-cadherin/catenin complexes.

p120 was originally identified as a substrate of pp60src and several receptor tyrosine kinases, but its function is not known. Recent studies revealed that this protein shows homology to a group of proteins, beta-catenin/Armadillo and plakoglobin (gamma-catenin), which are associated with the cell adhesion molecules cadherins. In this study, we examined whether p120 is associated with E-cadherin using the human carcinoma cell line HT29, as well as other cell lines, which express both of these proteins. When proteins that copurified with E-cadherin were analyzed, not only alpha-catenin, beta-catenin, and plakoglobin but also p120 were detected. Conversely, immunoprecipitates of p120 contained E-cadherin and all the catenins, although a large subpopulation of p120 was not associated with E-cadherin. Analysis of these immunoprecipitates suggests that 20% or less of the extractable E-cadherin is associated with p120. When p120 immunoprecipitation was performed with cell lysates depleted of E-cadherin, beta-catenin was no longer coprecipitated, and the amount of plakoglobin copurified was greatly reduced. This finding suggests that there are various forms of p120 complexes, including p120/E-cadherin/beta-catenin and p120/E-cadherin/plakoglobin complexes; this association profile contrasts with the mutually exclusive association of beta-catenin and plakoglobin with cadherins. When the COOH-terminal catenin binding site was truncated from E-cadherin, not only beta-catenin but also p120 did not coprecipitate with this mutated E-cadherin. Immunocytological studies showed that p120 colocalized with E-cadherin at cell-cell contact sites, even after non-ionic detergent extraction. Treatment of cells with hepatocyte growth factor/scatter factor altered the level of tyrosine phosphorylation of p120 as well as of beta-catenin and plakoglobin. These results suggest that p120 associates with E-cadherin at its COOH-terminal region, but the mechanism for this association differs from that for the association of beta-catenin and plakoglobin with E-cadherin, and thus, that p120, whose function could be modulated by growth factors, may play a unique role in regulation of the cadherin-catenin adhesion system.

Binding Sites↗
PubMed1994

Purification and characterization of nuclear factors binding to the negative regulatory element D of human apolipoprotein A-II promoter: a negative regulatory effect is reversed by GABP, an Ets-related protein.

We have previously shown that transcription of the human apolipoprotein A-II (apoA-II) gene is controlled by a complex set of regulatory elements [Cardot et al. (1993) Biochemistry 32, 9080-9093]. We have also identified previously described, as well as new activities which bind to these elements and influence to varying degrees the transcription of the apoA-II gene. DNA binding and competition assays indicated that element D binds three new activities, designated AIID1, AIID2, and AIID4, as well as an activity related to C/EBP. Activities AIID1, AIID2, and AIID4 were purified and characterized further in order to determine their function on the transcriptional regulation of human apoA-II gene. SDS-PAGE analysis as well as photoaffinity cross-linking of the affinity-purified AIID2 showed that it consists of three proteins with molecular masses ranging between 54 and 63 kDa. The amino acid sequence of tryptic peptides obtained from AIID2 protein bands revealed that it is homologous to GABP, an Ets-related protein. Similar analysis showed that affinity-purified AIID4 has an apparent molecular mass of 130 kDa. AIID1 activity was purified partially; in addition to binding to the apoA-II promoter, AIID1 also binds to the regulatory element C of apoCIII and may play a role in the transcriptional regulation of both genes. Methylation interference of G residues and permanganate modification of T residues indicated that the binding sites of AIID2 and AIID4 were contiguous on element D. However, the binding site of AIID1 overlaps with the binding sites of both AIID2 and AIID4. This suggests that the binding of AIID1 and AIID2 or of AIID1 and AIID4 may be mutually exclusive, whereas AIID2 and AIID4 may bind simultaneously. Transcription from a minimal promoter containing elements AB, C, and D of apoA-II increased 1.5- to 1.6-fold when element D is deleted, as well as by promoter mutations which eliminated the binding of both AIID1 and/or AIID4 to element D, but permitted the binding of AIID2/GABP. The findings suggest that element D has a negative regulatory role on apoA-II gene transcription when it is occupied by protein AIID1 and/or AIID4. This negative effect is reversed when element D is occupied only by the regulatory factor AIID2/GABP.

Amino Acid Sequence↗
PubMed2026

Genomic Profiling of Epidermal Growth Factor Receptor Mutation-Positive Non-Small Cell Lung Cancer after Progression on First-line Osimertinib: Phase II ORCHARD Study.

PURPOSE: Osimertinib is the standard of care for first-line treatment for epidermal growth factor receptor-mutated (EGFRm) non-small cell lung cancer (NSCLC). Understanding the tumor molecular profile of patients following progression on osimertinib could help inform optimal second-line treatment. PATIENTS AND METHODS: ORCHARD (NCT03944772), a phase II biomarker-directed study, enrolled patients with EGFRm NSCLC who progressed on first-line osimertinib to receive treatment based on their tumor molecular profile after progression. The study comprised three groups into which patients were allocated based on the molecular profile of their tumor, determined via next-generation sequencing (NGS) of a tumor biopsy. We report results from a prespecified, exploratory analysis of baseline tumor tissue and plasma samples to evaluate mechanisms of resistance to first-line osimertinib identified by tissue and plasma NGS. Agreement between tissue and plasma NGS data was also assessed. RESULTS: This study provided a comprehensive dataset exploring tissue (n = 400) and plasma (n = 191) genomics, enabling characterization of the histogenomic landscape after first-line osimertinib treatment. TP53 and MDM2/4 alterations were mutually exclusive and occurred in 86% of tumors. When combining tissue and plasma genomics, resistance alterations were detected in 87% of samples, with multiple resistance alterations in 46%. Alterations in the PI3K pathway, SOX2, and MYC were frequently detected in histologically transformed tumors. Additionally, differential patterns of co-occurring EGFR mutations in tumors with L858R versus exon 19 deletion were observed. CONCLUSIONS: This comprehensive analysis highlights potential heterogeneous resistance to first-line osimertinib treatment, providing a rationale for combining treatments with broad activity to improve patient outcomes. See related commentary by Gupta et al., p. 3718.

Humans↗
PubMed1992

Mutations altering substrate specificity in OHIO-1, and SHV-1 family beta-lactamase.

The OHIO-1 beta-lactamase does not normally hydrolyse oxyimino-beta-lactam substrates like cefotaxime, ceftriaxone, ceftazidime or aztreonam. We were able to select spontaneous mutants of an OHIO-1-bearing strain of Escherichia coli using the antibiotic substrates listed above by enrichment methods of frequencies of 10(-8)-10(-10) for all antibiotics except ceftazidime (frequency less than 10(-10)). Most mutants with increased resistance to the other beta-lactams were also more resistant to ceftazidime. Mutations identified by DNA sequencing included a Gly238----Ser238 substitution identical with the SHV-2 mutation previously described, cysteine and valine substitutions at the identical site, and a Gly242----Cys242 substitution. The Cys238 and Cys242 mutant enzymes had less affinity for aztreonam than had the other mutant enzymes. Hydrolysis of cefotaxime, but not cephaloridine, by the cysteine-substituted enzymes was inhibited by p-chloromercuribenzoate. The mutant enzymes had, in general, greater affinity for the mechanism-based inhibitors sulbactam, clavulanic acid and tazobactam. These results suggest two non-mutually exclusive hypotheses for the structural role of substitutions in this area of the enzyme. Either potential hydrogen-bond donors, such as serine and cysteine, interact directly with the beta-lactam molecules, or the steric bulk of these substitutions distorts the beta-pleated sheet such that the beta-lactam is held in a position favourable for stable binding and catalysis. Finally, our data raise questions about a strategy relying on oligonucleotide-probe technology to detect such mutations, because of the variety of substitutions that give rise to similar phenotypes.

Amino Acid Sequence↗
PubMed2006

Tying the loose ends together in DNA double strand break repair with 53BP1.

To maintain genomic stability and ensure the fidelity of chromosomal transmission, cells respond to various forms of genotoxic stress, including DNA double-stranded breaks (DSBs), through the activation of DNA damage response signaling networks. In response to DSBs as induced by ionizing radiation (IR), during DNA replication, or through immunoglobulin heavy chain (IgH) rearrangements in B cells of lymphoid origin, the phosphatidyl inositol-like kinase (PIK) kinases ATM (mutated in ataxia telangiectasia), ATR (ATM and Rad3-related kinase), and the DNA-dependent protein kinase (DNA-PK) activate signaling pathways that lead to DSB repair. DSBs are repaired by either of two major, non-mutually exclusive pathways: homologous recombination (HR) that utilizes an undamaged sister chromatid template (or homologous chromosome) and non- homologous end joining (NHEJ), an error prone mechanism that processes and joins broken DNA ends through the coordinated effort of a small set of ubiquitous factors (DNA-PKcs, Ku70, Ku80, artemis, Xrcc4/DNA lig IV, and XLF/Cernunnos). The PIK kinases phosphorylate a variety of effector substrates that propagate the DNA damage signal, ultimately resulting in various biological outputs that influence cell cycle arrest, transcription, DNA repair, and apoptosis. A variety of data has revealed a critical role for p53-binding protein 1 (53BP1) in the cellular response to DSBs including various aspects of p53 function. Importantly, 53BP1 plays a major role in suppressing translocations, particularly in B and T cells. This report will review past experiments and current knowledge regarding the role of 53BP1 in the DNA damage response.

Journal Article↗
PubMed1999

p53-mediated repression of alpha-fetoprotein gene expression by specific DNA binding.

Aberrant expression of the alpha-fetoprotein (AFP) gene is characteristic of a majority of hepatocellular carcinoma cases and serves as a diagnostic tumor-specific marker. By dissecting regulatory mechanisms through electromobility gel shift, transient-transfection, Western blot, and in vitro transcription analyses, we find that AFP gene expression is controlled in part by mutually exclusive binding of two trans-acting factors, p53 and hepatic nuclear factor 3 (HNF-3). HNF-3 protein activates while p53 represses AFP transcription through sequence-specific binding within the previously identified AFP developmental repressor domain. A single mutation within the DNA binding domain of p53 protein or a mutation of the p53 DNA binding element within the AFP developmental repressor eliminates p53-repressive effects in both transient-transfection and cell-free expression systems. Coexpression of p300 histone acetyltransferase, which has been shown to acetylate p53 and increase specific DNA binding, amplifies the p53-mediated repression. Western blot analysis of proteins present in developmentally staged, liver nuclear extracts reveal a one-to-one correlation between activation of p53 protein and repression of AFP during hepatic development. Induction of p53 in response to actinomycin D or hypoxic stress decreases AFP expression. Studies in fibroblast cells lacking HNF-3 further support a model for p53-mediated repression that is both passive through displacement of a tissue-specific activating factor and active in the presence of tissue-specific corepressors. This mechanism for p53-mediated repression of AFP gene expression may be active during hepatic differentiation and lost in the process of tumorigenesis.

Animals↗
PubMed1999

Evidence for transcriptional self-regulation of variable surface antigens in Paramecium tetraurelia.

Variable surface antigens are commonly found on free-living and parasitic protozoa, yet the regulation of antigen expression and switching is not fully understood in any system. A cell line of Paramecium tetraurelia stock 51 can express at least 11 different antigens yet only one type is found on the surface at any one time. Previous studies have shown that mutually exclusive expression of Paramecium surface antigens can be overcome if two antigen genes contain the same 5' coding region. In this article we utilize a gene chimera containing portions of A51 and B51 to analyze the effect of a frameshift mutation on transcription and steady-state mRNA levels. We show that a frameshift mutation near the 3' end prevents expression of the protein on the cell surface and reduces the rate of transcription of the corresponding gene. The difference in transcription is not the result of differences in plasmid copy number. We propose that expression of the antigen on the cell surface is part of a self-regulatory pathway that influences transcription of the corresponding gene. A model incorporating the previous and current data is presented.

Animals↗
PubMed2000

The nonsense-mediated decay pathway and mutually exclusive expression of alternatively spliced FGFR2IIIb and -IIIc mRNAs.

Exons IIIb and IIIc of the FGFR2 gene are alternatively spliced in a mutually exclusive manner in different cell types. A switch from expression of FGFR2IIIb to FGFR2IIIc accompanies the transition of nonmalignant rat prostate tumor epithelial cells (DTE) to cells comprising malignant AT3 tumors. Here we used transfection of minigenes with and without alterations in reading frame and with and without introns to examine how translation affects observed FGFR2 splice products. We observed that nonsense mutations in other than the last exon led to a dramatic reduction in mRNA that is abrogated by removal of downstream introns in both DTE and AT3 cells. The mRNA, devoid of both IIIb and IIIc exons (C1-C2), is a major splice product from minigenes lacking an intron downstream of the second common exon C2. From these observations, we suggest that repression of exon IIIc and activation of exon IIIb inclusion in DTE cells lead to the generation of both C1-IIIb-C2 and C1-C2 products. However, the C1-C2 product from the native gene is degraded due to a frameshift and a premature termination codon caused by splicing C1 and C2 together. Derepression of exon IIIc and repression of exon IIIb lead to the generation of both C1-IIIc-C2 and C1-C2 products in AT3 cells, but the C1-C2 product is degraded. The C1-IIIb-IIIc-C2 mRNA containing a premature termination codon in exon IIIc was present, but at apparently trace levels in both cell types. The nonsense-mediated mRNA decay pathway and cell type-dependent rates of inclusion of exons IIIb and IIIc result in the mutually exclusive expression of FGFR2IIIb and IIIc.

Alternative Splicing↗
PubMed1994

Bw4-reactive and Bw6-reactive antibodies recognize multiple distinct HLA structures that partially overlap in the alpha-1 helix.

Bw4 and BW6 epitopes are expressed by mutually exclusive sets of HLA-B alleles and some HLA-A and HLA-C alleles. To test whether antithetical structures are required to express Bw4 and Bw6 epitopes, we measured binding of Bw4-reactive and Bw6-reactive alloantibodies and mAbs to HLA-B7 variants. A triple substitution of HLA-B7 alpha-1 helix residues 80, 82, and 83 created Bw4 and destroyed Bw6 epitopes detected by alloantibodies and mAbs. Both Bw4-reactive and Bw6-reactive mAbs competed for binding to HLA-B7 variants with single substitutions at residues 82 and 83. Substitutions of residues H93 and D119 which form a salt bridge in HLA-A2 also permitted binding by both Bw4-reactive and Bw6-reactive mAbs, suggesting that Bw4 and Bw6 epitopes are conformationally dependent. Six Bw4-reactive mAbs showed four distinct patterns of binding to HLA-B7 variants. Detailed analysis of 74 HLA-B7 single-residue variants showed that Bw6-reactive SFR8-B6 binding was prohibited by mutations altering the distal end of the alpha-1 helix and the nearby connecting loop. In contrast, Bw6-reactive BB7.6 binding required both alpha-1 and alpha-2 helix residues. Thus, Bw4-reactive and Bw6-reactive Abs recognize multiple distinct HLA structures that partially overlap in the alpha-1 helix. As both Bw4 and Bw6 epitopes are expressed by some HLA-B7 variants, mutually exclusive expression of Bw4 and Bw6 epitopes in naturally occurring HLA class 1 molecules may reflect evolutionary pressure.

Antibodies, Monoclonal↗
PubMed2026

Disentangling the cellular composition of FLCN-mutated tumors in Birt-Hogg-Dubé Syndrome by spatial transcriptomics.

Birt-Hogg-Dubé (BHD) syndrome is a hereditary cancer predisposition syndrome caused by pathogenic variants in the folliculin (FLCN) gene and is associated with an increased risk of multifocal renal tumors. FLCN-mutated tumors (FMTs) often exhibit morphological heterogeneity with mixed morphological features resembling renal oncocytoma (RO) and chromophobe renal cell carcinoma (chRCC), yet the molecular basis underlying the heterogeneous morphologic features and the morphologic-genomic correlations remain poorly defined. In our prior work, we identified mutually exclusive expressions of L1 cell adhesion molecule (L1CAM) and forkhead box I1 tboxI1 (FOXI1) labeling the two morphologically distinct cellular populations in BHD-associated FMTs, leading to the hypothesis that these two tumor compartments may have distinct molecular features and may reflect different nephron epithelial differentiation states. In this follow-up study, we tested this hypothesis using L1CAM and FOXI1 as morphology-guided markers for spatial transcriptomic profiling of the distinct tumor compartments in FMTs with the NanoString GeoMX Digital Spatial Profiler (DSP). Six FMTs from three patients with BHD and three normal kidney tissues were analyzed. L1CAM+ and FOXI1+ area of interest (AOI) were collected from tumor areas with various tumor compositions, including L1CAM+ dominant, FOXI1+ dominant, and mixed tumor areas. Spatial transcriptomic analysis identified distinct gene expression signatures in L1CAM+ and FOXI1+ FMT compartments independent of the local tumor compositions. FOXI1+ tumor cells showed robust enrichment for intercalated cells (IC)-associated gene signatures. In contrast, L1CAM+ tumor cells exhibited a heterogeneous transcriptional profile, with partial overlap across a spectrum of renal tubular epithelial cell types rather than a definitive principal cell-like identity. Despite this compartment-specific differences, both compartments share expression of a panel of tumor signature genes, including glycoprotein nmb (GPNMB) gene, and a core of cancer related biological functions and signaling pathways. Together, these findings refined the prior dichotomous model of BHD-associated renal tumors and support a model in which L1CAM+ and FOXI1+ tumor compartments represent divergent evolutionary or differentiation states with a common FLCN-mutant neoplastic transcriptional program. This spatial transcriptomic profiling provides molecular evidence for the morphological heterogeneity of FMTs and insights on the tumor biology of BHD-associated FMTs.

Birt-Hogg-Dubé↗
PubMed1997

Phorbol ester-induced sensitisation of adenylyl cyclase type II is related to phosphorylation of threonine 1057.

Following up the results from previous studies on chemical fragmentation of TPA-treated, [32P]phosphate labeled adenylyl cyclase type II (AC II) (Böl, G. F., Hülster, A., and Pfeuffer, T. in press) we have replaced serine 871 or threonine 1057 by alanine using site directed mutagenesis. Both mutants had unimpaired catalytic activity, however enhancement by phorbolester TPA was reduced by 60-80 % in the T1057A mutant, but not in the S871A mutant. The stimulation of adenylyl cyclase type II by betagamma subunits of heterotrimeric G-pro teins and that by PKC have been previously shown to be mutually exclusive (Zimmermann and Taussig (1996), J. Biol. Chem. 271, 27161-27166). This is in line with the present findings that AC II expressed in COS-1 cells was only barely stimulated (10%) by coexpressed betagamma-subunits in presence of TPA. Mutation of threonine 1057 to alanine however caused partial regain of betagamma-stimulation in the presence of TPA by 40%, as compared to that of WT adenylyl cyclase type II which was 70% in the absence of TPA. These data strongly implicate the importance of threonine 1057 as phosphate acceptor following PKC-mediated sensitisation of adenylyl cyclase type II.

Adenylyl Cyclases↗
PubMed2003

The coat protein of prunus necrotic ringspot virus specifically binds to and regulates the conformation of its genomic RNA.

Binding of coat protein (CP) to the 3' nontranslated region (3'-NTR) of viral RNAs is a crucial requirement to establish the infection of Alfamo- and Ilarviruses. In vitro binding properties of the Prunus necrotic ringspot ilarvirus (PNRSV) CP to the 3'-NTR of its genomic RNA using purified E. coli- expressed CP and different synthetic peptides corresponding to a 26-residue sequence near the N-terminus were investigated by electrophoretic mobility shift assays. PNRSV CP bound to, at least, three different sites existing on the 3'-NTR. Moreover, the N-terminal region between amino acid residues 25 to 50 of the protein could function as an independent RNA-binding domain. Single exchange of some arginine residues by alanine eliminated the RNA-interaction capacity of the synthetic peptides, consistent with a crucial role for Arg residues common to many RNA-binding proteins possessing Arg-rich domains. Circular dichroism spectroscopy revealed that the RNA conformation is altered when amino-terminal CP peptides bind to the viral RNA. Finally, mutational analysis of the 3'-NTR suggested the presence of a pseudoknotted structure at this region on the PNRSV RNA that, when stabilized by the presence of Mg(2+), lost its capability to bind the coat protein. The existence of two mutually exclusive conformations for the 3'-NTR of PNRSV strongly suggests a similar regulatory mechanism at the 3'-NTR level in Alfamo- and Ilarvirus genera.

3' Untranslated Regions↗
PubMed2005

Mechanisms of Disease: genetic and epigenetic alterations that drive bladder cancer.

There is substantial evidence for the existence of mutually exclusive molecular pathways to tumorigenesis, in the formation of papillary and invasive carcinomas, respectively. The most common genetic alterations in low grade papillary transitional-cell carcinoma (TCC) are loss of heterozygosity of part or all of chromosome 9 and activating mutations of the fibroblast growth factor receptor 3 (FGFR3). The pathway to development of invasive TCC seems to start with dysplasia, progress to carcinoma in situ, followed by invasion of the lamina propria. The most frequent genetic alteration in dysplasia and carcinoma in situ is mutation of TP53, followed by loss of heterozygosity of chromosome 9. A marker for progression in TCC is loss of chromosome 8p, which occurs in approximately 60% of bladder tumors. Global trends of increased genomic instability and aberrant methylation of cytosine residues in DNA correlate with increased tumor invasion and progression. When researching markers of bladder cancer for clinical use, it is important that biomedical pathways and their alterations are measured in the same tumor populations. This review examines the published data and proposes a model for the mechanisms behind bladder cancer development.

Epigenesis, Genetic↗
PubMed2004

hnRNP A1 and the SR proteins ASF/SF2 and SC35 have antagonistic functions in splicing of beta-tropomyosin exon 6B.

Mutually exclusive splicing of exons 6A and 6B from the chicken beta-tropomyosin gene involves numerous regulatory sequences. Previously, we identified a G-rich intronic sequence (S3) downstream of exon 6B. This element consists of six G-rich motifs, mutations of which abolish splicing of exon 6B. In this paper, we investigated the cellular factors that bind to this G-rich element. By using RNA affinity chromatography, we identified heterogeneous nuclear ribonucleoprotein (hnRNP) A1, the SR proteins ASF/SF2 and SC35, and hnRNP F/H as specific components that are assembled onto the G-rich element. By using hnRNP A1-depleted HeLa nuclear extract and add-back experiments, we show that hnRNP A1 has a negative effect on splicing of exon 6B. In agreement with in vitro data, artificial recruitment of hnRNP A1, as a fusion with the MS2 coat protein, also represses splicing of exon 6B ex vivo. In contrast, ASF/SF2 and SC35 activate splicing of exon 6B. As observed with other systems, hnRNP A1 counteracts the stimulating effect of the SR proteins. Moreover, cross-linking experiments show that both ASF/SF2 and SC35 are able to displace binding of hnRNP A1 to the G-rich element, suggesting that the binding sites for these proteins are overlapping. These data indicate that the G-rich sequence is a composite element that acts as an enhancer or as a silencer, depending on which proteins bind to them.

Alternative Splicing↗
PubMed1998

Interaction of Agouti protein with the melanocortin 1 receptor in vitro and in vivo.

Agouti protein and Agouti-related protein (Agrp) are paracrine-signaling molecules that normally regulate pigmentation and body weight, respectively. These proteins antagonize the effects of alpha-melanocyte-stimulating hormone (alpha-MSH) and other melanocortins, and several alternatives have been proposed to explain their biochemical mechanisms of action. We have used a sensitive bioassay based on Xenopus melanophores to characterize pharmacologic properties of recombinant Agouti protein, and have directly measured its cell-surface binding to mammalian cells by use of an epitope-tagged form (HA-Agouti) that retains biologic activity. In melanophores, Agouti protein has no effect in the absence of alpha-MSH, but its action cannot be explained solely by inhibition of alpha-MSH binding. In 293T cells, expression of the Mc1r confers a specific, high-affinity binding site for HA-Agouti. Binding is inhibited by alpha-MSH, or by Agrp, which indicates that alpha-MSH and Agouti protein bind in a mutually exclusive way to the Mc1r, and that the similarity between Agouti protein and Agrp includes their binding sites. The effects of Agouti and the Mc1r in vivo have been examined in a sensitized background provided by the chinchilla (Tyrc-ch) mutation, which uncovers a phenotypic difference between overexpression of Agouti in lethal yellow (Ay/a) mice and loss of Mc1r function in recessive yellow (Mc1re/Mc1re) mice. Double and triple mutant studies indicate that a functional Mc1r is required for the pigmentary effects of Agouti, and suggest that Agouti protein can act as an agonist of the Mc1r in a way that differs from alpha-MSH stimulation. These results resolve questions regarding the biochemical mechanism of Agouti protein action, and provide evidence of a novel signaling mechanism whereby alpha-MSH and Agouti protein or Agrp function as independent ligands that inhibit each other's binding and transduce opposite signals through a single receptor.

Agouti Signaling Protein↗
PubMed1996

Association of a novel GTP binding protein, DRG, with TAL oncogenic proteins.

TAL1 is a basic helix-loop-helix (bHLH) protein involved in hematopoietic development. In T cell acute lymphoblastic leukemic cells, TAL1 is aberrantly overexpressed and is thought to contribute to oncogenesis. To identify proteins that interact with TAL1 in mediating leukemogenesis, we used TAL1 as a bait in a two-hybrid interaction screen, and isolated a cDNA clone that encodes a unique GTP binding protein, DRG. The interaction between DRG and TAL1 was confirmed both in vitro and in vivo. DRG was also shown to bind in vitro to two TAL1-related proteins, TAL2 and Lyl1. Mutational analyses showed that the HLH domain of TAL1 was necessary and sufficient for its interaction with the C-terminus of DRG. Furthermore, while DRG and E47 compete to interact with TAL1, TAL1 binds to DRG and E47 in a mutually exclusive manner. In rat embryonic fibroblast transformation assays, DRG stimulated the cotransforming activity of c-myc and ras. Based on these results, DRG appears to be a potential target for TAL-like oncoproteins.

Amino Acid Sequence↗