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PubMed2003

Identification and characterization of G beta 3s2, a novel splice variant of the G-protein beta 3 subunit.

The T-allele of a polymorphism (C825T) in the gene for the G-protein beta 3 subunit (GNB3) is associated with cardiovascular and metabolic disorders, distinct cellular features and altered drug responses. The molecular mechanisms that give rise to this complex phenotype have been linked to the occurrence of G beta 3s, a splice variant of GNB3. G beta 3s is predominantly expressed in cells with the 825T-allele. In the present study we describe the identification and characterization of an additional G beta 3 splice variant referred to as G beta 3s2. Its mRNA is expressed in heart, blood cells and tumour tissue, and its expression is also tightly associated with the GNB3 825T-allele. G beta 3s2 is generated by alternative splicing using non-canonical splice sites. G beta subunits belong to the family of propeller proteins and consist of seven regular propeller blades. Transcripts for G beta 3s2 are lacking 129 bp of the coding sequence of the wild-type G beta 3 protein. Thus the predicted structure consists of only six propeller blades, which resembles the structure of G beta 3s. Co-immunoprecipitation analyses indicated that G beta 3s2 dimerizes with different G gamma subunits, e.g. G gamma 5, G gamma 8(C) and G gamma 12. In Sf9 insect cells, expression of G beta 3s2 together with G gamma 12 enhances receptor-stimulated activation of G alpha(i2). Expression of G beta 3s2 in mammalian cells activated the mitogen-activated protein kinase cascade. Together, these results suggest that G beta 3s2 is a biologically active G beta variant which may play a role in the manifestation of the complex phenotype associated with the 825T-allele.

Alleles↗

PubMed1994

Identification and DNA sequence analysis of 15 new alpha 1-antitrypsin variants, including two PIQ0 alleles and one deficient PIM allele.

We have investigated the molecular basis of 15 new alpha 1-antitrypsin (alpha 1AT) variants. Phenotyping by isoelectric focusing (IEF) was used as a screening method to detect alpha 1AT variants at the protein level. Genotyping was then performed by sequence analysis of all coding exons, exon-intron junctions, and the hepatocyte-specific promoter region including exon Ic. Three of these rare variants are alleles of clinical relevance, associated with undetectable or very low serum levels of alpha 1AT:the PI*Q0saarbruecken allele generated by a 1-bp C-nucleotide insertion within a stretch of seven cytosines spanning residues 360-362, resulting in a 3' frameshift and the acquisition of a stop codon at residue 376; a point mutation in the PI*Q0lisbon allele, resulting in a single amino acid substitution Thr68(ACC)-->Ile(ATC); and an in-frame trinucleotide deletion delta Phe51 (TTC) in the highly deficient PI*Mpalermo allele. The remaining 12 alleles are associated with normal alpha 1AT serum levels and are characterized by point mutations causing single amino acid substitutions in all but one case. This exception is a silent mutation, which does not affect the amino acid sequence. The limitation of IEF compared with DNA sequence analysis, for identification of new variants, their generation by mutagenesis, and the clinical relevance of the three deficiency alleles are discussed.

Alleles↗

PubMed2005

Engineering of a thioglycoligase: randomized mutagenesis of the acid-base residue leads to the identification of improved catalysts.

Thioglycoligases are recently introduced variants of retaining glycosidases in which the acid-base catalyst has been mutated, rendering them capable of thioglycoside synthesis. The original acid-base mutant of Agrobacterium sp. beta-glucosidase (E170A) was previously shown to be an effective thioglycoligase carrying out glycosyltransfer from 2,4-dinitrophenyl glycosides to several different thio sugar acceptors. Here we report the generation of a screen for improved thioglycoligases, randomized mutagenesis of the acid-base catalyst E170 and identification of variants superior to E170A. Furthermore we have established a coupled assay allowing kinetic analysis of isolated variants and found that Abg E170Q is 5-fold faster than Abg E170A when 2,4-dinitrophenyl glucoside is used as donor and 100-fold faster when glucosyl azide is used. To demonstrate its utility, different acceptor and donor sugar combinations were employed to produce thio-linked di- or trisaccharides in high yields, showing the considerable versatility of the system for the synthesis of carbohydrate mimetics.

Acids↗

PubMed2002

Identification and characterization of a novel human cathepsin L splice variant.

Human cathepsin L (hCATL) has been implicated in a variety of physiological and pathological processes. It was hitherto known to be encoded by four mRNA species, namely hCATL A, AI, AII and hCATL B, differing in their 5' untranslated regions (UTRs). Of these, hCATL A, AI and AII are produced by the alternative splicing of the same primary transcript. HCATL AI and hCATL AII, lack 27 and 90 bases, respectively, from the 3' end of exon 1 of hCATL A. The present study describes the identification of a new splice variant hCATL AIII, which similarly lacks 145 bases from the 3' end of exon 1 of hCATL A. It is produced by the splicing out of 136-280 bases of the first exon in addition to intron 1 of hCATL A, which together serve as an intron for hCATL AIII. HCATL AIII was observed to be the most abundant splice variant in five different human cell lines. In vitro transcription coupled translation studies revealed that hCATL AIII is translated with 4.4-, 3.9- and 1.6-fold higher efficiency as compared to hCATL A, AI and AII, respectively. These results were further confirmed by measuring the enzymatic activities of the in vitro translated products. Cloning of hCATL AIII UTR upstream to luciferase reporter gene resulted in a 3.75-fold higher expression of the reporter gene as compared to the luciferase construct containing UTR of hCATL A. Thus, we have identified a novel human cathepsin L splice variant, hCATL AIII, which is most abundant in human cell lines and is translated with highest efficiency. Our results demonstrate either the presence of a positive or absence of a negative cis-acting regulatory element(s) in the UTR of hCATL AIII that is sufficient to confer translational advantage to a heterologous mRNA. The predominance of this most efficiently translated splice variant in malignant cells suggests that it plays a key role in the over-expression of human cathepsin L in cancer.

3T3 Cells↗

PubMed1993

Comparison of the protein and DNA approaches for the characterization of a beta-globin chain variant, hemoglobin Cocody [beta 21 (B3) Asp--->Asn], in a Caucasian patient.

Over the past few years, the methodologies used for the identification of hemoglobin A variants have been greatly improved. Both the protein- and DNA-based strategies have their own advantages and limitations. In this report we illustrate the use of both assays for the characterization of a hemoglobin Cocody variant in a women of Spanish descent. After evaluating the mobility value matrix of the abnormal hemoglobin, the amino acid transition was determined by HPLC and micro-sequencing of the protein. The beta 21 Asp was shown to be substituted by an Asn. At the DNA level, the only nucleotide replacement responsible for this amino acid substitution is GAT--->AAT at codon 21. The analysis of the beta-globin gene by denaturing gradient gel electrophoresis (DGGE) method showed that the mutation was situated in a fragment including exon 1. The hemoglobin variant was then identified to be hemoglobin Cocody by DNA sequencing of this fragment.

Adult↗

PubMed2006

Identification of a novel human tissue factor splice variant that is upregulated in tumor cells.

Tissue factor (TF) is a transmembrane glycoprotein that serves as the prime initiator of blood coagulation and plays a critical role in thrombosis and hemostasis. In addition, a variety of tumor cells overexpress cell-surface TF, which appears to be important for tumor angiogenesis and metastasis. To elucidate the mechanism involved in the upregulation of TF in human tumor cells, a comprehensive analysis of TF mRNA from various normal and tumor cells was performed. The results of these studies indicate that, in addition to possessing a normal full-length TF transcript and minor levels of an alternatively spliced transcript known as alternatively-spliced tissue factor (asTF), human tumor cells express additional full-length TF transcripts that are also generated by alternative splicing. Reverse transcriptase-polymerase chain reaction (RT-PCR) and 5'-rapid amplification of cDNA ends- (5'-RACE) based analyses of cytoplasmic RNA from normal and tumor cells revealed that there is alternative splicing of the first intron between exon I and exon II resulting in 2 additional TF transcripts. One of the transcripts has an extended exon I with inclusion of most of the first TF intron (955 bp), while the second transcript is formed by the insertion of a 495 bp sequence, referred to as exon IA, derived from an internal sequence of the first intron. The full length TF transcript with alternatively spliced novel exon IA, referred to as alternative exon 1A-tissue factor (TF-A), represented approximately 1% of the total TF transcripts in normal cells, but constituted 7-10% of the total TF transcript in tumor cells. Quantitative real-time RT-PCR analysis indicated that cultured human tumor cells contain 10-25-fold more copy numbers of TF-A in comparison to normal, untransformed cells. We propose that high-level expression of the novel TF-A transcript, preferentially in tumor cells, may have utility in the diagnosis and staging of a variety of solid tumors.

Adenocarcinoma↗

PubMed2002

HHV-8 subtypes in South Africa: identification of a case suggesting a novel B variant.

Human herpesvirus 8 (HHV-8) has been identified as the causative agent for all forms of Kaposi's sarcoma and is also associated with the development of body cavity-based B-cell lymphomas and multicentric Castleman's disease. HHV-8 genomes are now classified into five major subtypes (A-E) that reflect sequence heterogeneity in the highly variable open reading frame (ORF) K1. To identify HHV-8 subtypes associated with different forms of Kaposi's sarcoma, we compared the ORF 26 and ORF-K1 gene sequences from South African patients with the prototype strains of the major subtypes, as well as published sequences from other African strains. DNA prepared from Kaposi's sarcoma biopsies and/or peripheral blood lymphocytes were available from 14 patients with postrenal transplant (iatrogenic) Kaposi's sarcoma, six patients with the African endemic form, and one patient with AIDS-related body cavity-based B-cell lymphoma. We identified a B2 subtype in six patients, four of whom also had a novel B5 type ORF 26 polymorphism. Two patients had B2 type patterns for both the ORF 26 and ORF-K1 genes. The ORF-K1 subtype A5 was identified in samples from three patients with a B3/C2 type polymorphism in the ORF 26 gene. A novel ORF-K1 B variant strain was identified in a patient with African endemic Kaposi's sarcoma, who also had a B3/C2 class ORF 26 pattern. In 58.3% of iatrogenic Kaposi's sarcoma patients, a B5-type ORF 26 gene sequence pattern was identified. No association was found among particular subtypes, geographical origin of patients, or clinical presentation.

Adult↗

PubMed1996

Identification of novel exon-deleted progesterone receptor variant mRNAs in human breast tissue.

Using an approach based on the co-amplification of wild-type and exon deleted progesterone receptor (PR) variant cDNAs, we identified exon-deleted PR variant mRNAs in both normal and neoplastic human breast tissues. Several naturally occurring variants, whose sequences revealed precise whole exon deletions, may encode putative PR-like proteins which lack some functional domains of the wild-type PR molecule. We suggest that these PR variant proteins could have a pathophysiological role in progestin action, as suggested for estrogen receptor variant proteins.

Breast↗

PubMed1989

Identification of virus neutralizing epitopes on naturally occurring variants of type A12 foot-and-mouth disease virus.

Four naturally occurring antigenic variants of foot-and-mouth disease virus type A12 were examined for their capacity to be neutralized by a number of monoclonal antibodies (MAb) which recognize different sites on the virus surface. The VP1 coding region of the RNA genome was sequenced and amino acid changes were determined for the variants. One of the neutralizing sites accounted for the differing antigenic properties of the variants and the epitope was mapped to amino acid residues 150-156 of VP1. Another epitope originally thought to occupy a single site in the area of amino acids 168-179 of VP1 was found on all of the variants. Competitive binding assays did not identify the exact binding site for this monoclonal antibody and the results suggest that the epitope may represent a site more complex than a sequential epitope. A polyclonal antiserum to a 13 kDa fragment of VP1 (residues 55-179) was found to have all the virus reactivity associated with sites located between residues 133-164 of VP1. In contrast, an antiserum to VP1 was found to have additional virus binding sites outside of the 133-164 region of VP1.

Antibodies, Monoclonal↗

PubMed2005

Identification of an N-terminal TRPC2 splice variant which inhibits calcium influx.

TRPC2 is a member of the transient receptor potential (TRP) superfamily of Ca2+-permeable channels expressed in nonexcitable cells. TRPC2 is involved in a number of physiological processes including sensory activation of the vomeronasal organ, sustained Ca2+ entry in sperm, and regulation of calcium influx by erythropoietin. Here, a new splice variant of TRPC2, called "Similar to mouse TRPC2" (smTRPC2), was identified consisting of 213 amino acids, largely coincident with the N-terminus of TRPC2 clone 17. This splice variant lacks all six TRPC2 transmembrane domains and the calcium pore. Expression of smTRPC2 was found in all tissues examined by RT-PCR and in primary erythroid cells by RT-PCR and Western blotting. Confocal microscopy of CHO-S cells transfected with TRPC2 clone 14 and smTRPC2 demonstrated that TRPC2 clone 14 and smTRPC2 both localize at or near the plasma membrane and in the perinuclear region. Cell surface localization of TRPC2 was confirmed with biotinylation, and was not substantially affected by smTRPC2 expression. Coassociation of TRPC2 c14 and alpha with smTRPC2 was confirmed by immunoprecipitation. To examine the functional significance of smTRPC2 expression, a CHO-S model was used to study its effect on calcium influx stimulated by Epo through TRPC2. Single CHO-S cells which express transfected Epo-R were identified by detection of green fluorescent protein (GFP). Cells that express transfected TRPC2 c14 or alpha were identified by detection of blue fluorescent protein (BFP). [Ca]i was quantitiated with Fura Red fluorescence using digital video imaging. Epo stimulated calcium influx through TRPC2 isoforms c14 and alpha, which was inhibited by coexpression of smTRPC2. These data demonstrate that a short splice variant of TRPC2 exists in many cell types, which associates with and modifies the activity of functional TRPC2 splice variants.

Alternative Splicing↗

PubMed2004

Identification of a Shiga-toxin type I variant containing an IS1203-like element, from Shiga-toxin producing Escherichia coli O157:H7.

We found two Shiga toxin producing Escherichia coli O157:H7 strains isolated from humans carrying the stx(1) gene with an IS1203-like element (designated as IS1203v(1)). The IS1203v(1) was inserted into the coding region of the A subunit 7 bp upstream from the TGA termination codon, resulting in a loss of two amino acid residues (Ser-Ser) from its C terminus. Toxicity of the Stx1 was confirmed by Vero cell assay. IS1203v(1) hardly affected the stx(1) gene in either its expression or the toxicity of its product.

Animals↗

PubMed2004

Identification and characterization of a human smad3 splicing variant lacking part of the linker region.

Smad3 is one of the signal transducers that are activated in response to transforming growth factor-beta (TGF-beta). We have identified and characterized a splicing variant of smad3. The splicing variant (smad3-Delta3) lacks exon 3 resulting in a truncated linker region. We could detect mRNA expression of smad3-Delta3 in all investigated human tissues. Real-time PCR analyses demonstrated that the fraction of smad3-Delta3 mRNA compared to normal smad3 varies between tissues. The amount of spliced mRNA was estimated to represent 0.5-5% of the normal smad3 mRNA. When smad3-Delta3 is overexpressed in a fibrosarcoma cell line, the Smad3-Delta3 is translocated to the nucleus upon TGF-beta stimulation and binds the Smad responsive element. Using a CAGA luciferase reporter system, we demonstrate that Smad3-Delta3 has transcriptional activity and we conclude that Smad3-Delta3 possesses functional transactivating properties.

Alternative Splicing↗

PubMed2006

Identification of somatic and germline mitochondrial DNA sequence variants in prostate cancer patients.

Prostate cancer (PCa) is the second most frequent cancer among men in the European Union and the most common in the United States. Older age and a positive family history of PCa are important risk factors, but little is known about the disease aetiology. Mitochondria are involved in essential cellular pathways, some of which have been associated with tumorigenesis. We analysed the presence of sequence variants, depletion and rearrangements in the mitochondrial DNA (mtDNA) of PCa patients. Sequencing of the D-loop and genes RNR1 and 2, ND3, ND4L and ND4, and long-range and real-time PCR techniques were carried out on 51 samples (41 from patients and 10 from controls). Normal, hyperplastic or tumour samples were obtained from 17 patients. Six patients' seminal vesicles were also investigated as an additional patient's control tissue (these structures seldom develop tumours). Neither depletion nor mtDNA rearrangements were detected. In contrast, 94 mtDNA sequence variants were identified, 9 previously unreported. The regions presenting more sequence variants were MT-DLOOP (52%), MT-RNR2 (14%) and MT-ND4 (13%). The patients' seminal vesicles studied showed the same set of variants as the corresponding prostate, suggesting either that the pathogenic role of these particular variants is minor or that they participate in the prostatic carcinogenesis in combination with other factors absent in seminal vesicles. Five patients (29.4%) harboured eight somatic changes in the mtDNA. One affects a conserved residue and three have not been previously described. The analysis of other genes in the mtDNA molecule might demonstrate an even higher incidence of mtDNA somatic variants in these PCa patients.

Aged↗

PubMed2005

Identification and characterization of six new alternatively spliced variants of the human mu opioid receptor gene, Oprm.

The mu opioid receptor plays an important role in mediating the actions of morphine and morphine-like drugs. Receptor binding and a wide range of pharmacological studies have proposed several mu receptor subtypes, but only one mu opioid receptor (Oprm) gene has been isolated. Like the mouse and rat, the human Oprm gene undergoes alternative splicing. In the present studies, we have identified and characterized six new splice variants from the human Oprm gene using a reverse transcription-polymerase chain reaction strategy, yielding a total of 10 human splice variants of the mu opioid receptor MOR-1. All the variants identified contained exons 1, 2 and 3, but differed from MOR-1 itself and each other by splicing downstream from exon 3, resulting in different amino acid sequences. Northern blot analysis demonstrated expression of the variant mRNAs. Receptor binding assays established that these variants belonged to the mu opioid receptor family with limited differences in mu opioid ligand affinities and selectivity. However, adenylyl cyclase and [35S]GTPgammaS binding assays revealed major differences in both potency and efficacy among these variants. The dissociation between binding affinity, potency and efficacy for the opioids among these variants may provide insights into the wide range of opioid responses among these agents observed clinically and opens new avenues in designing selective drugs based upon their efficacy and potency rather simple binding affinity.

Adenylyl Cyclases↗

PubMed2025

Identification of a novel RBCK1 splice site donor variant in Basset Hounds with glycogen storage disease myopathy.

Glycogen storage diseases (GSDs) are rare, typically inherited, disorders caused by various defects in glycogen metabolism enzymes, generally resulting in the accumulation of glycogen in several tissues. Recently, two young adult Basset Hound (BH) littermates were diagnosed with GSD via postmortem histopathology, with excess glycogen manifesting in both cardiac and smooth muscle. Using whole genome sequencing, a homozygous splice site donor variant was identified in exon 8 of RBCK1, a gene which encodes an E3 ubiquitin ligase, in both littermates, suggesting an autosomal recessive mode of inheritance. The presumptive loss of the splice site donor is predicted to result in premature termination in the mid-domain of the protein. Screening for the variant in related (n = 21) and unrelated (n = 124) BHs identified one additional affected littermate and nine familial heterozygous carriers. No variant alleles were present in the unrelated BH population, establishing the novelty of the identified mutation. RBCK1 variants have previously been associated with polyglucosan body myopathy type 1 (PGBM1), a type of GSD characterized by skeletal muscle myopathy, cardiomyopathy, and polyglucosan accumulation in humans. To date, no reported variants in RBCK1 have been identified in dogs or other large animals associated with GSD, making this the first naturally occurring large animal model of PGBM1 due to an RBCK1 defect.

Animals↗

PubMed2001

Identification and tissue distribution of novel KET/p63 splice variants.

The human p53 protein family comprises three members - p53, p63 and p73. Whereas only one p53 variant is known multiple isoforms of p63 and p73 have been described. Depending on the isoform p63 influences p53-responsive genes in a p53-like or -distinct manner. We have cloned multiple splice variants of keratinocyte transcription factor (KET), the rat ortholog of human p63. Several tissue specific variations of exon 1 resulting in different amino-terminal ends were identified. Transactivation properties of the splice variants inversely correlated with the length of the N-termini as determined by activation of the p53-responsive p21 promotor. Multiple KET isoforms are colocalized in different rat tissues. The amino-terminal truncated form DeltaNKETalpha is expressed in epithelial tissues, while expression of the most p53-like KET isotype TAKETgamma was detected in skeletal muscle. Expression of a major KET variant appears to be a cell-type specific rather than a differentiation specific phenomenon.

Alternative Splicing↗

PubMed2001

Identification and tissue distribution of two differentially spliced variants of the rat carnitine transporter OCTN2.

In this paper we show that the only known Na(+) dependent transporter of carnitine in mammals, organic cation transporter number 2 (OCTN2), is subject to differential splicing. Cloning of OCTN2 in different rat tissues identified two splicing variants. We have developed a real time quantitative polymerase chain reaction method for quantification of these splice variants. Both splice variants could be detected in all tissues examined with a relative abundance of 0.1-1% of the full length transcript. We also draw attention to the previously described mutations in clinical examples of primary carnitine deficiency in humans where the described mutations appear to be those of a splicing or mis-splicing event.

Alternative Splicing↗

PubMed2002

Identification of a tachykinin NK(2) receptor splice variant and its expression in human and rat tissues.

The tachykinins substance P, neurokinin A and neurokinin B are implicated in different diseases and play an important role in neuroimmunomodulation. These peptides interact with three distinct types of tachykinin receptors termed NK(1), NK(2) and NK(3). While most mammalian genes encoding G protein-coupling cell membrane receptors are intron-less, the three tachykinin receptors contain introns in their genomic structures. In the present study, we have identified a splice variant of the tachykinin NK(2) receptor that results from skipping of exon 2 in the processing of the tachykinin NK(2) receptor mRNA. By using reverse transcription-polymerase chain reaction analysis, we observed that the tachykinin NK(2) receptor splice variant, that we named NK(2)beta, appeared in different human and rat tissues that also express the wild type, tachykinin NK(2)alpha isoform. Compared to tachykinin receptor NK(2)alpha isoform mRNA levels, the NK(2)beta isoform was strongly expressed in human and rat uteri, expressed in a moderate degree in the rat urinary bladder, colon, duodenum and stomach and unexpressed in the rat cerebral cortex, kidney, thoracic aorta, skeletal muscle and heart. These data describe the first known tachykinin receptor splice variant and suggest that the variety of tachykinin receptors may be further expanded through the generation of splicing isoforms. The presence of the truncated isoform may have a physiological significance in the regulation of tachykinin NK(2) receptor protein levels.

Adult↗