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PubMed2005

Multiple variants of the ING1 and ING2 tumor suppressors are differentially expressed and thyroid hormone-responsive in Xenopus laevis.

The tumor suppressor candidate, inhibitor of growth (ING) is implicated in the control of apoptosis, cell cycle progression, chemosensitivity, and senescence. There are at least five different genes in mammals, ING1-ING5, and there is limited evidence that multiple transcript variants exist for ING1 that encode proteins with different functions. No variants have yet been reported for other ING genes. Here, we report the isolation of seven Xenopus laevis (x)ING1 and three xING2 transcript variants and give the first evidence for their independent regulation by thyroid hormone (TH). Comparison with mammalian genes reveals conservation in gene structure. xING1 and xING2 transcript variants are differentially expressed in adult tissues with the greatest number of variants expressed at high levels in brain, testis, and eye. During metamorphosis of the tadpole into a frog, the hindlimb, tail, and brain undergo growth, apoptosis, or remodeling, respectively. We show that xING1 and xING2 transcript variants are significantly reduced in the hindlimb while many variants increase in the tail. These transcript variants remain largely unchanged in the brain during this developmental period. By exposing premetamorphic tadpoles to TH, a precocious metamorphosis is induced. We identify specific variants whose steady state levels are significantly affected by TH at 24 and 48h of exposure. Although several of the variants show expression patterns reminiscent of that observed in natural metamorphosis, the results indicate that additional factors may be involved to influence the steady state transcript levels during development.

Animals↗

PubMed2002

Expression of novel splice variants of the G protein subunit, Go alpha, is tissue-specific and age-dependent in the rat.

Heterotrimeric G proteins play an essential role in coupling numerous surface membrane receptors to intracellular signal transduction pathways. Relatively little is known about the splice variants of G proteins, including whether they undergo differential expression as a function of aging. We screened for splice variants of the alpha subunit of the dominant inhibitory G protein family member G(o) in a variety of tissues from rat and examined the expression of these splice variants during development. The splice variants were characterized using specific primers for G(o)alpha1 and G(o)alpha2 in conjunction with reverse transcription-polymerase chain reaction, and subsequently sequenced. G(o)alpha1 expression dominated over G(o)alpha2 in all neuronal tissues screened, including cerebral cortex, pituitary, spinal cord, colon myenteric plexus, dorsal root ganglion, and prenatal cortex. The sequence data of G(o)alpha1 supports the presence of three splice variants: G(o)alpha1a, G(o)alpha1b, G(o)alpha1c. The G(o)alpha1a variant was reported previously [J. Biol. Chem. 262 (1987) 14241], whereas G(o)alpha1b and G(o)alpha1c represent novel variants. The G(o)alpha1b splice variant demonstrates a 94 bp deletion using a cryptic donor site in exon 10. The G(o)alpha1c variant demonstrates a complete deletion of exon 10. A protein product with a molecular weight of approximately 34 kDa consistent with that expected for G(o)alpha1c was identified using Western blot analysis and two-dimensional gel electrophoresis. The expression of G(o)alpha1a decreased postnatally, supporting a potential physiological role during fetal development, whereas G(o)alpha1c expression increased postnatally. The age-dependent and tissue-specific expression of the G(o)alpha1 splice variants presage a broader functional role than has been observed historically with G(o).

Aging↗

PubMed1998

Study of the expression of the genetic variants of human alpha1-acid glycoprotein in healthy subjects using isoelectric focusing and immunoblotting.

Human alpha1-acid glycoprotein (AAG) exists as an heterogeneous population of two or three genetic variants (ORM1 F1 and/or S and ORM2 A) in the plasma of most individuals. The ORM1 and ORM2 variants have a separate genetic origin. AAG belongs to the acute-phase proteins, which, under conditions of inflammation, increase several-fold in concentration. Additionally, there is evidence to suggest that it is not only the concentration but also the distribution of the two gene products of AAG (ORM1 and ORM2) that alter in such conditions. Variations of the relative concentrations of the AAG variants in certain diseases, such as cancer, can only be shown by reference to data collected in healthy people. In this study, we have investigated a group of 74 healthy subjects (42 men and 32 women) for AAG concentrations, AAG phenotypes and relative proportions of genetic variants in plasma. The specific assay of AAG was carried out by an immunonephelometric method and the phenotyping was performed, after desialylation of AAG, by analytical isoelectric focusing. Detection of the AAG variants was made by immunoblotting and their relative proportions were determined by laser densitometry analysis. The AAG plasma concentrations in the healthy group ranged between 0.28 and 0.92 g/l (mean value 0.50+/-0.14 g/l). The relative proportions of the variants derived from the two genes of AAG were variable, depending on the individual, but the amount of ORM1 variants almost always exceeded that of the ORM2 variant. No sex-related differences were observed in respect either in the total AAG level nor the relative proportions of the ORM1 and ORM2 variants. The data collected in this study may serve as a reference towards the investigation of possible changes in the expression of the genetic variants of AAG in chronic inflammatory diseases.

Adult↗

PubMed1998

Changes in expression and microheterogeneity of the genetic variants of human alpha1-acid glycoprotein in malignant mesothelioma.

Human alpha1-acid glycoprotein (AAG), an acute-phase plasma protein, is heterogeneous in the native state and polymorphic in the desialylated state. The AAG heterogeneity is mainly explained by a variable glycan chain composition in its five glycosylation sites. The AAG polymorphism is due to the presence of genetic variants. Three main variants are observed for AAG, ORM1 F1, ORM1 S and ORM2 A, which have a separate genetic origin. In this paper, we have used different isoelectric focusing (IEF) methods and chromatography on immobilized metal affinity adsorbent to study the relative occurrence of the genetic variants of AAG in relation to changes in microheterogeneity, in plasma and pleural effusions of patients with malignant mesothelioma (MM). The results were compared to those obtained with the variants in plasma of healthy individuals. Significant changes in variant distribution were observed in the MM samples, that corresponded to a rise in the proportion of the ORM1 variants and a fall in that of the ORM2 variant. However, the concentration in MM plasma increased for both variants. The AAG in MM plasma and effusion fluids was found to be more heterogeneous on IEF than AAG of healthy plasma. The evidence of stronger concentrations of both the high and low pI forms of AAG in the MM samples suggested two kinds of changes in charge heterogeneity. These two changes were shown to be attributed to different variants--i.e. the high pI forms to ORM1 F1 and S and the low pI forms to ORM2 A, after fractionation of AAG by chromatography on immobilized copper(II) ions. These results indicate specific changes in both the expression and glycosylation for each AAG variant, according to its separate genetic origin, in MM.

Aged↗

PubMed1997

A novel estrogen receptor variant mRNA lacking exons 4 to 6 in breast carcinoma.

A novel estrogen receptor mRNA splice variant which lacks the entire exon 5 and part of exon 4 and 6 was identified using reverse transcription PCR in human breast carcinomas. The variant was translated in vitro and produced a protein of approximately Mr 31000 which lacked the ligand binding domain. The binding of the variant estrogen receptor (ER) to a synthetic estrogen-responsive element (ERE) was compared with that of the wild-type ER (wtER). The variant ER bound weakly to the synthetic ERE, both in the presence and absence of estradiol, whilst the wtER bound strongly in the absence and the presence of estradiol. When wtER and variant ER were simultaneously translated in vitro, no heterodimerization was observed using band shift assay. Addition of increasing amounts of variant ER protein to the wtER in the ERE binding reaction showed that the variant protein competed with the binding of the wtER to the synthetic ERE. Furthermore, variant ER are not transcriptionally active. The variant was also expressed in 96% of the 102 breast tumours analysed, of which 62 were tamoxifen-resistant tumours. The expression of this variant was significantly higher (relative to ER) in untreated ER-positive breast tumours which were both progesterone receptor (PgR) negative and pS2 negative phenotype.

Amino Acid Sequence↗

PubMed2003

Human CYP1A1 allelic variants: baculovirus expression and purification, hydrodynamic, spectral, and catalytical properties and their potency in the formation of all-trans-retinoic acid.

Three human cytochrome P450 1A1 (CYP1A1) allelic variants, namely wild-type (CYP1A1.1), CYP1A1.2 (I462V), and CYP1A1.4 (T461N), were expressed as C-terminal His-tagged fusions including a thrombin cleavage site in Spodoptera frugiperda insect cells by baculovirus infection. The variants were expressed with 30-90 nmol (1.8-5.4 mg) spectrally active cytochrome P450 per one liter of culture and purified to electrophoretic homogeneity by Ni-agarose chromatography. The recombinant variants were structurally characterized by UV/Vis, ultracentrifugation, and EPR. Optical and EPR spectra showed all three variants predominantly in high spin state; moreover, EPR indicated changes in the electronic structure of the heme iron of the two mutant variants. Sedimentation equilibrium experiments demonstrated the purified variants in dimeric state in the presence of 0.2% emulgen+0.05% cholate. Higher detergent concentration, the presence of imidazole, and cleavage of the His-tag led to monomerization. Catalytic activity of all purified variants was reconstituted with purified human NADPH-P450 reductase and dilaurylphosphatidylcholine. Enzyme kinetics of ethoxyresorufin O-deethylation revealed similar K(m) ( approximately 0.4 microM) for all variants but slightly different V(max) values (CYP1A1.1: 4.2, CYP1A1.2: 7.0, and CYP1A1.4: 3.0 nmol/min/nmol CYP1A1). The extended C-terminus influenced the enzymatic activity only slightly. All three variants are able to produce significant amounts of all-trans-retinoic acid from all-trans-retinal with V(max) of 4.0, 3.3, and 5.6 nmol/min/nmol CYP1A1 and K(m) values of 111, 83, and 250 microM for CYP1A1.1, CYP1A1.2, and CYP1A1.4, respectively. Availability of the three purified human CYP1A1 variants should facilitate further characterization of their role in metabolism of endogenous and exogenous compounds as well as structural studies.

Alleles↗

PubMed1995

Trans effects on cysteine ligation in the proximal His93Cys variant of horse heart myoglobin.

Three variants of horse heart myoglobin (Mb) in which the proximal His93 residue has been replaced with a Cys residue have been constructed and studied by NMR, EPR, and MCD spectroscopy to evaluate the contributions of proximal and distal residues to the coordination environment of the heme iron in these proteins. Although no experimental conditions were identified that allowed quantitative ligation of the cysteine residue to the heme iron in the His93Cys variant, all of the spectroscopic evidence collected for the His93Cys/His64Ile and His93Cys/His64Val double variants supports the assignment of thiolate as the ligand to iron in the oxidized forms of these variants. The double metMb variants exhibit Soret maxima that are considerably blue-shifted, 1H NMR spectra with decreased mean methyl resonances, and EPR spectra with highly rhombic g values. These spectroscopic data for the Fe(III) variants resemble the corresponding properties reported for ferricytochrome P-450. The decrease in the reduction potential of the double variants by 280 mV relative to wild-type protein is also consistent with the low midpoint potential of cytochrome P-450. MCD spectroscopy of these variants confirms that the proximal cysteine residue is not bound in the reduced forms of these proteins and, in the case of the His93Cys variant, that the distal histidine is coordinated to the iron. Similar coordination environments were created in the ferrimyoglobin variants by cyanogen bromide modification, which resulted in cyanation of the sulfur atom and prevented the ligation of Cys93 to the heme iron.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1998

Tenascin-cytotactin (TN-C) variants in pseudophakic/aphakic bullous keratopathy corneas.

PURPOSE: To examine pseudophakic/aphakic bullous keratopathy (PBK/ABK) human corneas for patterns of expression of tenascincytotactin (TN-C) variants known to mediate specific cellular functions, viz. anti-adhesion (high molecular mass (M(r))) and adhesion (low/intermediate M(r)). METHODS: PBK/ABK corneas were selected to encompass only those with bullae and without inflammation, scarring or neovascularisation. Serial sections from these and normal corneas were labelled with antibodies BC-4 (recognising all TN-C variants) and BC-2 (specific for the high M(r) TN-C variant). Bound antibody was revealed with an avidin-biotin peroxidase technique. In a given pair of corneal sections, positivity with BC-4 but not BC-2 indicates localisation of low/ intermediate M(r) TN-C variants and absence of the high M(r) TN-C variant. BC-2 identifies the high M(r) variant. RESULTS: There was no immunostaining with either BC-2 or BC-4 in normal corneas except at the corneoscleral interface, where both BC-2 and BC-4 were immunolocalised. In PBK/ABK corneas, BC-2 staining was seen in 5 of 13 corneas and was restricted mainly to epithelial basement membrane (BM) overlying bullae. BC-2 did not label the stroma. BC-4 immunostaining was present in all PBK/ABK corneas and was localised in epithelial BM, both epithelial and stromal borders of bullae, pannus, endothelial BM and in oedematous stromal regions. CONCLUSIONS: TN-C variants are differentially expressed in PBK/ABK corneas. The high M(r) variant is restricted mainly to epithelial BM overlying bullae, while low/intermediate M(r) variants occur in epithelial BM, both epithelial and stromal borders of bullae, and in pannus. Given the in vitro functions of TN-C, a role for promoting epithelial dehiscence and reattachment to the substratum in PBK/ABK corneas by high and low/intermediate M(r) variants respectively is likely.

Aged↗

PubMed2026

Hemizygous loss-of-function variants of EIF1AX are associated with a syndromic neurodevelopmental disorder.

Pathogenic variants of genes encoding initiation factors can cause neurological diseases, including neurodevelopmental disorders and brain abnormalities. The eukaryotic translation initiation factor 1 A, X-linked (EIF1AX) is a gene located at Xp22.12 that plays an important role in the regulation of translation initiation. Here, we identified de novo hemizygous EIF1AX variants in male individuals with neurodevelopmental disorders and explored their possible involvement in these neurological disorders. We performed trio-based exome or whole genome sequencing in four families. The pathogenicity of EIF1AX variants was evaluated using a molecular dynamic simulation and transgenic Drosophila models. We identified four de novo hemizygous EIF1AX variants in four male individuals with variable neurodevelopmental delay, dysmorphic features, behavioral problems, ophthalmological abnormalities, and structural abnormalities in the brain. One variant was predicted to cause a splicing alteration, and minigene analysis confirmed exon skipping leading to the generation of a premature termination codon. In transgenic Drosophila harboring wild-type (WT) EIF1AX or the three other EIF1AX missense variants, overexpression of WT and the p.(Asn17Asp) variant caused structural abnormalities in the compound eye, whereas the p.(Lys64Glu) and p.(Asp90Gly) variants significantly reduced these eye abnormalities. In addition, WT overexpression resulted in significant axonal toxicity in the Drosophila optic nerve, causing a significant reduction in the number of axons, whereas all mutants showed only a mild reduction in axonal number. Our findings indicated that all variants resulted in different degrees of EIF1AX loss-of-function. Overall, EIF1AX is a novel gene for which loss-of-function variants appear to produce syndromic neurodevelopmental disorders in males.

Humans↗

PubMed2026

Targeted reflex RNA sequencing for enhanced variant classification on exome and genome sequencing improves patient outcomes.

RNA sequencing (RNA-seq) has been utilized to provide functional evidence regarding the impact of splicing variants. This study explores the utility of targeted reflex RNA-seq to inform classification of predicted splicing variants identified through clinical exome sequencing (ES) and genome sequencing (GS). A retrospective analysis was conducted on consecutive ES/GS cases completed at a single center in which targeted reflex RNA-seq was performed following identification of eligible variants. There were 131 cases (4.1%) that had at least one RNA-seq eligible variant reported, with eight of these cases having two unique eligible variants. Of the 139 eligible variants, 125 were classified as variants of uncertain significance (VUS). Sixty-four cases had targeted reflex RNA-seq completed with 27 cases having at least one variant reclassified (42.2%). After reclassification, 23 cases had positive results, and two cases had a likely diagnosis of an autosomal recessive condition. Clinical outcomes data regarding positive RNA-seq cases showed that 71% (10/14) had clinical management changes and 43% (6/14) had treatment changes. Incorporation of targeted reflex RNA-seq analysis into the diagnostic pipeline of rare diseases enhances variant classification and resolves uncertainty regarding predicted splice variants, leading to an estimated 1.6% increase in diagnostic yield of clinical ES/GS.

Journal Article↗

PubMed2002

Human melanocortin 1 receptor (MC1R) gene variants alter melanoma cell growth and adhesion to extracellular matrix.

Pigmentation is a significant determinant of individual susceptibility to cutaneous melanoma, with fair skinned subjects at highest risk of developing this neoplasm. Melanocortin 1 receptor (MC1R) gene variants alter pigment synthesis in vivo, and are causally associated with red hair and fair skin in humans. MC1R variants are more frequent in subjects with melanoma, and increase the risk of developing this tumour in sporadic and familial cases. MC1R variants may predispose to melanoma as a result of alterations in skin pigmentation (which affords less protection against incident ultraviolet radiation). However, melanoma cells synthesize and release alpha-melanocyte stimulating hormone (alphaMSH, the ligand for MC1R), therefore MC1R variants could alter the autocrine effects of alphaMSH on melanoma cell behaviour, thereby affecting early melanoma development and progression via non-pigmentary mechanisms. B16G4F melanoma cells, which are functionally null at Mc1r, were stably transfected with wild type and variant (Arg151Cys, Arg160Trp, and Asp294His) human MC1R. At similar MC1 receptor numbers per cell, alphaMSH increased intracellular cAMP in wild type MC1R transfected melanoma cells, but the cAMP response was compromised in the variant MC1R transfected clones. In growth inhibition experiments, alphaMSH significantly reduced growth of wild type MC1R transfected cells, but had no effect on cells transfected with variant MC1R. In addition, binding to fibronectin was significantly reduced by alphaMSH in the wild type transfectants whereas this was not observed in the variant transfected clones; binding to laminin was not affected by alphaMSH in this cell line. These results provide evidence for differences in melanoma cell behaviour secondary to MC1R variants, and suggest an alternative non-pigmentary mechanism whereby MC1R variants could modify melanoma susceptibility or progression.

Base Sequence↗

PubMed2005

Splice variants of the cyclic nucleotide phosphodiesterase PDE4D are differentially expressed and regulated in rat tissue.

Cyclic nucleotide PDE4 (phosphodiesterase 4) inhibitors are being developed as potent anti-inflammatory drugs for use in chronic lung diseases, but the complexity of the PDE4 family has hampered this process. The four genes comprising the PDE4 family, PDE4A, PDE4B, PDE4C and PDE4D, are all expressed as multiple splice variants. The most widely used criterion to identify PDE4 variants expressed endogenously is their migration on SDS/PAGE. However, when a PDE4D3-selective antibody was used for immunoprecipitation, the pattern of expression obtained did not confirm the expression predicted by SDS/PAGE. This observation, together with the recent discovery of additional PDE4D transcripts, prompted us to re-evaluate the pattern of expression of these variants. The nine rat PDE4D splice variants, PDE4D1 to PDE4D9, were cloned, their electrophoretic properties compared, and their in vivo mRNA and protein levels determined. Using this approach, we found that the pattern of distribution of the PDE4D splicing variants is more complex than previously reported. Multiple variants co-migrate in single immunoreactive bands, and variant-selective antibodies were necessary to discriminate between splice variants. Tissues that were thought to express only PDE4D3, express three closely related proteins, with PDE4D8 and PDE4D9 as the predominantly expressed forms. In addition, activation of cAMP signalling produces phosphorylation and activation of variants other than PDE4D3, and expression of PDE4D mRNA does not always correlate with the pattern of protein expression. As PDE4 inhibitors have different affinities for distinct PDE4D splicing variants, our results indicate that a better definition of the pattern of PDE4 expression is required for target validation.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2002

Wild-type and 'a' epitope variants in chronic hepatitis B virus carriers positive for hepatitis B surface antigen and antibody.

AIMS: This study aims to examine the genomic variants of the 'a' epitope in chronic hepatitis B virus (HBV) carriers positive for both hepatitis B surface antigen (HBsAg) and antibody to HBsAg (anti-HBs). METHODS: Eighteen HBV carriers were studied. Hepatitis B virus (HBV) DNA was extracted and the 'a' epitope region was amplified and sequenced. RESULTS: Eighteen Chinese asymptomatic HBV carriers were studied. There were 13 patients who were positive for both HBsAg and anti-HBs. Of these, one patient had only wild-type HBV, three had a viral mixture, and five had only 'a' epitope variant HBV. Of the three patients with a viral mixture, all had variants in the less conserved region (123-137). Of the five patients with pure HBsAg mutants, three had variants in the less conserved region while two had variants in the highly conserved region. In this study with a limited number of patients, the serum alanine aminotransferase (ALT) levels were higher in patients with wild-type HBV, compared with those with either 'a' epitope variants or a viral mixture consisting of wild type and variants. CONCLUSION: Eight of the nine (89%) patients positive for both HBsAg and anti-HBs harbored an 'a' epitope variant. The lower ALT levels seen in patients who had either pure 'a' epitope variant or a mixture of wild type and mutants suggest that a closer monitoring of these 'a' epitope variants should be required, as patients carrying these infectious viral strains may remain asymptomatic.

Adult↗

PubMed1979

Antigenic drift in type A influenza virus: peptide mapping and antigenic analysis of A/PR/8/34 (HON1) variants selected with monoclonal antibodies.

Variants of A/PR/8/34 (HON1) influenza virus, having hemagglutinin molecules with probably a single altered antigenic determinant, were isolated by growing the virus in the presence of the monoclonal hybridoma antibody PEG-1. The variants were analyzed by peptide mapping and characterized antigenically by using PEG-1 and four other monoclonal hybridoma antibodies to PR8 hemagglutinin. Peptide maps of the large hemagglutinin polypeptide, HA1, from 8 out of 10 variants showed a single changed peptide. This peptide from two of the variants was analyzed, and in each case a serine residue in the wild-type hemagglutinin was replaced by leucine in the variant. Although these eight variants showed identical peptide maps, one could be discriminated antigenically from the others with one of the hybridomas. (The peptide maps represented about one-third of the HA1 molecule.) Of the other two variants, one gave the same HA1 map as the wild type, but could be distinguished antigenically from wild-type virus by two of the hybridomas. The other was unique, and could be distinguished, both antigenically and by peptide mapping, from the other variants. Since a large number of the variants selected with PEG-1 showed the same peptide change, it is likely that this alteration in amino acid sequence (serine to leucine) was responsible for the inability of the variants to bind PEG-1 monoclonal antibody. We do not know, however, whether the changed amino acids were located within the antigenic sites or whether the change occurred somewhere else in the hemagglutinin molecule and altered the determinants through conformational changes.

Amino Acids↗

PubMed1987

Selection and characterization of T-cell variants lacking molecules involved in T-cell activation (T3 T-cell receptor, T44, and T11): analysis of the functional relationship among different pathways of activation.

A clone of the interleukin 2-producing Jurkat leukemia cell line termed JA3 (surface phenotype, T3+, Ti+, T44+, T11+, T40+) has been used to induce and select cell variants lacking surface molecules involved in T-cell activation. Following 200 rad of gamma-radiation (1 rad = 0.01 Gy), cells were treated with monoclonal antibodies (mAbs) directed to T3, Ti, T44, or T11 antigen and complement. After growth of the residual cells in culture, "negative" cells were cloned under limiting conditions. Depending on the specificity of the mAb used for the immunoselection, three groups of variants were obtained. (i) The use of mAbs directed to T3 or Ti resulted in cell variants that expressed the T3 Ti- T44+ Leu1+ T11+ T40+ 4F2+ HLA class I+ surface phenotype. (ii) Immunoselection with anti-T44 mAb resulted in 2 variants that shared the T3- Ti- T44- Leu1- T11+ T40+ 4F2+ HLA class I+ phenotype. (iii) Cell treatment with anti-T11 mAb resulted in 15 variants characterized by the lack of T11 antigen expression and of all the other T-cell-specific surface antigens. Therefore, it appears that the different sets of JA3 cell variants, like T cells at discrete stages of intrathymic differentiation, may follow a coordinated expression of surface differentiation antigens. Analysis of the functional responsiveness of the three distinct groups of JA3 cell variants to different stimuli showed that all produced interleukin 2 in response to A23187 calcium ionophore plus phorbol 12-myristate 13-acetate. The first group of variants (T3- Ti-) did not respond to stimulation with anti-T3, anti-Ti, or anti-T44 mAbs. Eight of 9 did not respond to phytohemagglutinin either; however, all responded to appropriate stimulatory combinations of anti-T11 mAbs (and to calcium ionophore). The second group of variants (T3-, Ti-, T44-, T11+), similar to the first group, did not respond to anti-T3, anti-Ti, anti-T44 mAbs, and phytohemagglutinin, but they were fully responsive to anti-T11 mAb. The last group of variants (lacking all the T-cell-specific surface antigens) only responded to calcium ionophore A23187.

Antibodies, Monoclonal↗

PubMed1996

Organization and alternate splicing of the murine folylpolyglutamate synthetase gene. Different splice variants in L1210 cells encode mitochondrial or cytosolic forms of the enzyme.

The organization of the murine folylpolyglutamate synthetase (FPGS) gene has been determined by sequence analysis that also revealed an interesting complexity in the case of exon 1. The entire nucleotide sequence of the L1210 FPGS cDNA, the 3'- and 5'-untranslated regions, the mitochondrial leader sequence, and the coding region were found to be distributed on 15 exons with an overall length of 10.358 kilobases. Two splice variants of exon 1 were identified by screening of an L1210 cell cDNA library. Variant I (exons 1a + 1b plus 2-15) incorporates all of the sequence homologous to the recently reported (Taylor, S. M., Freemantle, S. J., and Moran, R. G. (1995) Cancer Res. 55, 6030-6034) human exon 1, including two ATG start codons at positions +1 and +126, and encodes both mitochondrial and cytosolic form of FPGS. The most prevalent variant, Variant II (exons 1b plus 2 to 15), incorporates only a portion (92 nucleotides at the 3' end) of this sequence, incorporates only one ATG start codon at position +126, and encodes only cytosolic FPGS. The existence of this variant is consistent with the identification of an appropriately situated internal donor/acceptor site in what was believed to be exon 1. A third related variant (Variant III) with a novel 5' termini was originally identified by screening of a mouse liver cDNA library. This variant, which occurs at moderately low frequency in the L1210 cell cDNA library, incorporates an alternate to exon 1a (exon 1c) spliced to exon 1b plus exons 2-15 and encodes a different mitochondrial leader peptide than Variant I. The identification of these variants suggests another possible mechanism, i.e. at the level of precursor mRNA splicing, for regulating synthesis of mitochondrial versus cytosolic forms of FPGS in the cell. Exon 1c is positioned in the gene upstream of exon 1a separated by an intron of 56 nucleotides within a region of DNA sequence that like the homologous human sequence is distinctly promoter-like. However, the sequence of this region differs from the human sequence in terms of the number, position, and type of putative regulatory elements, particularly in regard to the number of SP-1 binding sites and the position of multiple transcription start sites as determined by enzymatic primer extension.

Alternative Splicing↗

PubMed1996

Suppression of prostaglandin E receptor signaling by the variant form of EP1 subtype.

A cDNA clone of prostaglandin (PG) E receptor EP1 subtype (rEP1) was isolated from a rat uterus cDNA library. It encodes 405 amino acid residues with seven transmembrane-spanning domains and couples to Ca2+ mobilization. In addition, three cDNA clones encoding a variant form of rEP1 were isolated. The open reading frame can code a 366-amino acid protein carrying a specific change of 49 amino acids from the middle of transmembrane segment VI to COOH terminus; it possesses a transmembrane segment VII-like structure lacking an intracellular COOH-terminal tail. Southern blot analysis of rat genomic DNA and genomic polymerase chain reaction demonstrated that these cDNAs were derived from a single copy gene. Northern blot analysis and ribonuclease protection assay revealed that both rEP1 and rEP1-variant receptor mRNAs were highly expressed in the kidney. Immunoblot with an antibody directed toward the specific region of rEP1-variant receptor showed that rEP1-variant receptor protein was expressed in the membrane of the kidney and Chinese hamster ovary (CHO) cells transfected with rEP1-variant cDNA. Thus, the rEP1-variant receptor is translated from mRNA which is not spliced at nucleotide position 952 in the segment VI transmembrane region. rEP1-variant receptor retained the ligand binding activity with affinity and specificity similar to rEP1 receptor, but lost the coupling of signal transduction systems by itself. However, when rEP1-variant receptor was stably co-expressed with rEP1 receptor in CHO cells, the Ca2+ mobilization mediated by EP1 receptor was significantly suppressed. Furthermore, when rEP1-variant receptor was expressed in CHO cells, cAMP formation by activation of endogenous EP4 receptor was strongly blocked. These results suggest that the rEP1-variant receptor may affect the efficiency of signal coupling of PGE receptors and attenuate the action of PGE2 on tissues.

Amino Acid Sequence↗

PubMed2002

Spatially distributed alternative splice variants of the renal Na-K-Cl cotransporter exhibit dramatically different affinities for the transported ions.

Three splice variants of the renal Na-K-Cl cotransporter (NKCC2 F, A, and B) are spatially distributed along the thick ascending limb of the mammalian kidney. To test whether NKCC2 splice variants differ in ion transport characteristics we expressed cDNAs encoding rabbit NKCC2 F, A, and B in Xenopus oocytes and determined the ion dependence of bumetanide-sensitive (86)Rb influx. The three splice variants of NKCC2 showed dramatic differences in their kinetic behavior. The medullary variant F exhibited 3-4-fold lower affinity than variants A and B for Na(+) and K(+). Chloride affinities also markedly distinguish the three variants (K(m)F = 111.3, K(m)A = 44.7, and K(m)B = 8.9 mm Cl(-)). Thus, the kinetic properties of the NKCC2 splice variants are consistent with the spatial distribution of the variants along the thick ascending limb as they are involved in reabsorbing Na(+), K(+), and Cl(-) from a progressively diluted fluid in the tubule lumen. Variant B also showed an anomalous inhibition of rubidium influx at high extracellular Na(+) concentrations, possibly important in its highly specialized role in the macula densa. The adaptation of the kinetic characteristics of the NKCC2 variants to the luminal concentrations of substrate represents an excellent example of functional specialization and diversity that can be achieved through alternative mRNA splicing.

Alternative Splicing↗