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At least 667 records · Page 37Linked to original sources
PubMed1996

Systematic screening for mutations in the human serotonin-2A (5-HT2A) receptor gene: identification of two naturally occurring receptor variants and association analysis in schizophrenia.

A statistically significant association between a silent mutation (102T/C) in the serotonin-2A (5-HT2A) receptor gene and schizophrenia has recently been reported in a sample of Japanese patients and healthy controls. This finding suggests that genetic predisposition to schizophrenia may be affected by a functional 5-HT2A receptor variant that is in linkage disequilibrium with 102T/C. In the present study, we have sought to identify genetic variation in the 5-HT2A receptor gene by screening genomic DNA samples from 91 unrelated subjects comprising 45 patients with schizophrenia and 46 healthy controls by using single-strand conformation analysis. We have identified four nucleotide sequence variants. Two sequence changes would result in protein alterations: a substitution of threonine by asparagine at position 25 (Thr25Asn), and a substitution of histidine by tyrosine at position 452 (His452Tyr). In order to test for a possible contribution to the development of schizophrenia, we have determined allele frequencies in extended samples of unrelated patients and healthy controls. The two amino acid substitutions are found with similar frequencies in patients and controls, indicating that the presence of these variants is not causally related to the development of schizophrenia. However, the reported association of the non-coding polymorphism 102T/C with the disease has also been detected in our sample (P=0.041, odds ratio=1.28, 95% confidence interval 1.012-1.623).

Alleles↗
PubMed2000

Identification and characterization of naturally occurring variants of the macrophage scavenger receptor (SR-A).

The scavenger receptor (SR) family comprises a group of cell surface proteins functionally defined by their ability to bind chemically modified lipoproteins. In macrophages, the class A Type I and Type II SRs (SR-AI/II) are thought to play a key role in adherence to and phagocytosis of infectious agents. Immunoprecipitation studies show that the rat anti-SR-AI/II monoclonal antibody 2F8 detects the mature, trimeric form of the receptor expressed in peritoneal macrophages from A/J, but not from C57Bl/6J (B6) mice. Subsequent sequencing of cDNA and genomic clones indicates that SR-AI and AII of A/J and B6 mice differ in sequence at nine positions, two in the cytoplasmic domain and seven in the extracellular spacer and alpha-helical coiled coil domains. These sequence polymorphisms are non-conservative and produce distinct receptor molecules that differ by four charged residues and alter recognition of the receptor by the monoclonal 2F8 antibody. The B6 SR-AI/II haplotype appears unique, since most inbred strains analyzed show the A/J-type haplotype. Interestingly, several of the B6 polymorphic variant residues are conserved in human and bovine receptors, suggesting a recent divergence of the A/J haplotype. Initial studies in CHO-derived cells expressing individual receptor isoforms indicate that the A/J and B6 receptors are stable and can mature into oligomers expressed in the membrane fractions of these cells. In these transfectants, no major functional differences were detected between receptors of the two haplotypes with respect to internalization and degradation of (125)I-labeled acetylated LDL. However, since SR-AI/II recognizes a large number of structurally unrelated anionic molecules, the possibility that different haplotypes may affect either binding and release of other ligands, or receptor recycling, cannot be excluded.

Animals↗
PubMed1998

Identification of brain-specific splicing variants of the hDLG1 gene and altered splicing in neuroblastoma cell lines.

The human homologue of Drosophila tumor suppressor dlg, hDLG1, is one of the proteins known to interact with APC, a tumor suppressor for colorectal cancer. Alternative splicing of this gene generates transcripts either with [insertion 1 (I1)] or without 99 nucleotides in the 5' part of the dlg homology repeats (DHR) domain. We found almost equivalent expression of these two splicing variants in most human tissues; however, in skeletal muscle the transcript with the 99-bp insertion was predominant, and in the brain, that without the 99-bp insertion was expressed predominantly. We also examined alternative splicing in the region between the SH3 and GUK domains where two different sizes of insertions, 34 nucleotides (I2) or 100 nucleotides (I3), had been reported, and found various splicing patterns among the tissues examined. In brain we detected six different, alternatively spliced transcripts, two of which included a novel, 36-bp, brain-specific exon encoding a peptide bearing significant homology to a portion of rat synapse-associated protein, SAP97/PSD95. Subsequently, we investigated the splicing patterns of the hDLG1 gene in 24 neuroblastoma cell lines. In two-thirds of these lines, the splicing patterns were altered from those observed in normal brain tissue. As one-third retained the normal brain-splicing pattern, the loss of normal splicing of hDLG1 may not in itself cause formation of tumors, but it might reflect the biological character of individual neuroblastomas.

Adaptor Proteins, Signal Transducing↗
PubMed1989

Identification of a deoxyribonucleic acid allelic variant for beta 1-4 galactosyltransferase expression associated with male sperm binding/penetration infertility.

Studies on mouse sperm-egg binding and fertilization have been suggested to involve the interaction of sperm-associated beta 1-4 galactosyltransferase with egg zona pellucida glycoproteins. A population of human males, whose sperm demonstrated an inability to penetrate ovulated zona pellucida-free hamster eggs in vitro, were examined for the level of activity of beta 1-4 galactosyltransferase. The level of enzyme activity was found to be reduced in human sperm isolated from this group of individuals compared with a known hamster penetration-positive group. Analysis of the deoxyribonucleic acid from these individuals by Southern hybridization with a putative human complementary deoxyribonucleic acid clone to beta 1-4 galactosyltransferase identified a unique allele lacking 0.8 and 0.4 kb restriction fragments on digestion with the endonuclease Taq I. These results represent the first evidence to suggest that mutations could be associated with the human gene for galactosyltransferase. Our data help to clarify one of the possible molecular mechanisms responsible for sperm-egg binding/penetration interactions.

Alleles↗
PubMed1996

Identification of cyclic AMP-phosphodiesterase variants from the PDE4D gene expressed in human peripheral mononuclear cells.

To determine whether the expression of different PDE4D variants is unique to the rat or conserved through evolution, we have characterized the different PDE4D mRNAs expressed in human peripheral blood mononuclear cells. RT-PCR was performed using primers based on rat sequences and mRNAs from mononuclear cells. The specifically amplified fragments had a size identical to that predicted for rat PDE4D1, PDE4D2 and PDE4D3. Sequencing confirmed that these fragments are derived from the human PDE4D gene. Their sequence was highly homologous to that reported for the rat variants. cDNAs corresponding to the entire ORF of human PDE4D2 and PDE4D3 were expressed in mammalian cells, causing a large increase in PDE activity. Western blot analysis of human peripheral blood mononuclear cell extracts demonstrated the presence of proteins corresponding to the recombinant PDE4D1 and PDE4D2. The pattern of splicing and different promoter usage of the PDE4D gene is therefore conserved during evolution, which indicates an important physiological role.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1995

Identification of a new apolipoprotein E variant (E2 Arg142-->Leu) in type III hyperlipidemia.

A new rare apolipoprotein E mutant was identified as we were investigating the apolipoprotein E genotype of patients with type III hyperlipidemia (HLP III). The unusual DNA restriction fragment length polymorphism profile and then the sequence analysis of a PCR amplified fragment of the proband's apo E gene revealed a simple base substitution (G-->T) at nucleotide 3836. This mutation leads to the replacement of arginine by leucine at position 142 of the mature protein. The proband carried the mutant allele at the heterozygous status with an epsilon 3 allele. Subsequently, analysis of the proband's father's apo E gene showed that same mutated allele associated with an epsilon 2 allele. The two subjects presented a dysbetalipoproteinemia in which this new apo E variant could be implicated.

Aged↗
PubMed1993

An immunoblotting procedure for screening glycophorins and band 3 protein in the same blots. Identification of glycophorin and band 3 variant forms.

An immunoblotting procedure is described which makes it possible to screen multiple blood samples for the presence of glycophorin and band 3 variant forms with altered electrophoretic mobility. The procedure can be simplified by using whole red blood cell hemolysates instead of membranes for SDS-polyacrylamide gel electrophoresis. The use of hemolysates also has the advantage that antigens sensitive to proteolysis are not degraded in vitro. The same nitrocellulose blots were used for immunoenzymatic detection of glycophorins with a set of anti-glycophorin monoclonal antibodies, and for autoradiographic detection of band 3-derived bands with 125I-labeled anti-band 3 monoclonal antibody. The screening of 157 Caucasian blood samples revealed the presence of a slower-migrating form of band 3 in seven cases and variant glycophorin in one case. The variant glycophorin exhibited the features of hybrid glycophorin of B-A type.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1995

Molecular cloning of a cDNA for the human inositol 1,4,5-trisphosphate receptor type 1, and the identification of a third alternatively spliced variant.

The Inositol 1,4,5-trisphosphate receptor (IP3R) is an intracellular calcium channel involved in coupling cell membrane receptors to calcium signal transduction pathways within the cell. We have cloned a cDNA for the human type 1 inositol 1,4,5-trisphosphate receptor. The sequence contains the S2 splice site which appears to be the region most divergent between rat and human. We now report an additional alternatively spliced region in the coupling domain, that is 9 amino acids long, which we term S3. Alternatively spliced forms are found in both human and rat. PCR analysis of brain and peripheral tissues from human and rat shows both transcripts of the type 1 inositol 1,4,5-trisphosphate receptor in all tissues. The long form predominates in most brain regions (except the cerebellum) while the short form predominates in peripheral tissues. The sequence of the longer form in human appears to create an additional consensus protein kinase C phosphorylation site.

Alternative Splicing↗
PubMed1989

Compartmentation of alpha-tubulin in neurons: identification of a somatodendritic-specific variant of alpha-tubulin.

The composition of alpha-tubulin in axons was compared with that in cell bodies and dendrites in the rat. Pure axons and highly enriched preparations of cell bodies and dendrites were obtained from explant cultures of sympathetic neurons, dissolved directly into sodium dodecyl sulfate, and analysed by isoelectric focusing gel electrophoresis. Immunoblots using two different monoclonal antibodies against alpha-tubulin and fluorographs of [35S]methionine- and [3H]tyrosine-labeled alpha-tubulin all show several variants common to both compartments, and an additional variant, more basic than the others, prominent in the somatodendritic compartment but absent from the axonal compartment. This variant, termed alpha s, partitions with both cytoskeletal and soluble tubulin pools, indicating that microtubules in the somatodendritic compartment differ from microtubules in the axonal compartment with respect to alpha-tubulin composition. Pulse-chase and immunoprecipitation analyses suggest that extensive post-translational modification of alpha-tubulin occurs in the neuron. Newly synthesized alpha-tubulin (10-min pulse-label) is relatively simple in profile, consisting of a major band with the mobility of alpha s, and a minor band slightly lower in isoelectric point. The profile is somewhat more complex after 5 h chase time, but is much more complex after 20 h. The generation of additional bands appears to be at the expense of the band co-migrating with alpha s, suggesting that alpha s is the unmodified form of a primary translation product that undergoes extensive post-translational modification. The unmodified form of the primary translation product is restricted to the somatodendritic compartment, while many of its derivatives are present throughout the neuron.

Animals↗
PubMed1991

The search for south European cystic fibrosis mutations: identification of two new mutations, four variants, and intronic sequences.

The major mutation in the cystic fibrosis (CF) gene is a 3-bp deletion (delta F508) in exon 10. About 50% of the CF chromosomes in Southern Europe carry this mutation, while other previously described mutations account for less than 4%. To identify other common mutations in CF patients from the Mediterranean area, we have sequenced, exon by exon, 16 chromosomes that did not show the delta F508 deletion from a selected panel of eight unrelated CF patients. We describe here one missense and one nonsense mutation, and four sequence polymorphisms. We have also found two previously reported mutations in three chromosomes. Overall, these mutations may account for about 20% of CF alleles in the Italian and Spanish populations. No other mutations were detected in 10 out of 16 CF chromosomes after analyzing about 90% of the coding region of the CF gene, and 39 out of 54 intron/exon boundaries. Therefore, about 26% of CF mutations remain to be identified. In addition we provide the intron/exon boundary sequences for exons 4 to 9. These results together with previously reported linkage data suggest that in the Mediterranean populations further mutations may lie in the promoter region, or in intron sequences not yet analyzed.

Base Sequence↗
PubMed2006

Identification of a truncated alternative splicing variant of human PPARgamma1 that exhibits dominant negative activity.

We have identified a novel variant of human peroxisome proliferator-activated receptor gamma (hPPARgamma), derived from insertion of a novel exon 3'. Insertion leads to the introduction of a premature stop codon, resulting in the formation of a truncated splice variant of PPARgamma1 (PPARgamma1(tr)). Western blot analysis confirmed the presence of PPARgamma1(tr) in tumor-derived cell lines. Although PPARgamma1(tr) interfered with transcriptional activity of wild-type PPARgamma1 (PPARgamma1(wt)), activity could be rescued by cotransfection with a vector expressing p300. Overexpression of PPARgamma1(tr) protein in CHO cells greatly enhanced their proliferation and anchorage-independent colony growth on soft agar. These data demonstrate that PPARgamma1(tr) is an important physiologic isoform of PPARgamma that modulates cellular functions of PPARgamma1(wt).

Alternative Splicing↗
PubMed2006

Identification and expression of alternative splice variants of the mouse Ppp1r3b gene in lung epithelial cells.

Hepatic glycogen-targeting subunit GL of protein phosphatase-1 (PP1), which is encoded by the gene Ppp1r3b, is one of a family of proteins that target PP1 to glycogen and regulate its activity on enzymes of glycogen metabolism. GL is primarily expressed in the liver where it promotes hepatic glycogen synthesis in response to insulin. Here, we show that GL is highly expressed in embryonic mouse lungs. GL utilizes two alternative promoters and 5' non-coding exons, which produce at least three alternatively spliced transcripts that encode identical proteins. Expression of GL in the lung is detectable as early as embryonic day (E) 12.5 and increases by E16.5, however, it declines before birth. In situ hybridization of mouse embryonic lung revealed that GL is expressed in bronchial epithelial cells. Thus, the temporal and spatial expression of GL in the lung suggests that it has a potential role in glycogen metabolism during lung development.

Alternative Splicing↗
PubMedDate not supplied

Identification and characterization of a novel variant in the highly conserved catalytic center of Rab11a.

Small GTPases of the Rab family regulate vesicular traffic and distribution of proteins in different cell types. Rab11a is a member of this GTP hydrolyzing protein class and acts as a mediator of insulin stimulated translocation of the glucose transporter GLUT4 in peripheral tissues including heart and skeletal muscle. Here we report on Rab11a Q70R, a mutation in the catalytic center of Rab11a, observed in the cardiomyoblast cell line H9c2. Analysis of GTPase activity showed that Rab11a Q70L acts as a classical constitutive active mutant. Interestingly, the GTPase activity of Rab11a Q70R was not significantly different from the enzymatic activity of the Rab11a Q70 wild type protein. We therefore conclude that the glutamine residue of Rab11a at position 70 is not strictly essential for GTPase activity of this protein in contrast to Ras and other Rab proteins.

Animals↗
PubMed2006

Identification and characterization of four splicing variants of ovine POU1F1 gene.

Expression of POU1F1 gene, a member of the POU homeodomain family of transcription factors, is necessary for normal differentiation, development and survival of three anterior pituitary cell types (thyrotrophs, somatotrophs and lactotrophs) and for the proper expression of growth hormone (GH), prolactin (PRL), thyroid-stimulating hormone (TSH) genes and POU1F1 gene itself. Alternative splicing forms of this gene have been reported in different species, with few functional studies. Apart from the POU1F1-Wild-type with the expected length, in this work we isolated three additional splicing variants: POU1F1-beta, with a 78 bp insert in the trans-activation domain; POU1F1-gamma that lacks exon 3 and POU1F1-delta that lacks exons 3, 4 and 5. Four different protein isoforms were also detected by Western blot in the sheep pituitary tissue. Functional assays were performed to study the trans-activation of GH and PRL promoters by the splicing variants. Regarding the PRL promoter, the beta variant presented only 12% of the Wild-type trans-activation capacity. Variants gamma and delta showed no capacity to trans-activate PRL promoter. Both gamma and delta variants acted as repressors of Wt, reducing significantly the trans-activation made by Wt alone (p<0.05). Concerning the GH promoter, the beta variant presented a trans-activation capacity 10% higher than Wt. Wt and beta variants strongly interact in the activation of GH promoter doubling the trans-activation potential of Wt. Variants gamma and delta showed no capacity to trans-activate the GH promoter and both acted as repressors, reducing significantly (p<0.001) the trans-activation performed by Wt. This work presents, for the first time, the characterization of four splicing forms of Ovis aries POU1F1 gene.

Alternative Splicing↗
PubMed2003

Evaluation of a line probe assay for identification of hepatitis B virus precore variants in serum from chronic hepatitis B carriers.

A prototype line probe assay (LiPA) for identifying hepatitis B virus (HBV) precore variants (INNO-LiPA HBV precore) was evaluated using a panel of 50 sera from 46 patients with HBV infection. The assay detected sequence variations detected commonly in the precore promoter region and in amino acid codons 28 and 29 of the precore gene. There was strong agreement between INNO-LiPA HBV precore results and those of a codon 28 point mutation assay (PMA), with identical results obtained in 40 of 43 sera (93%) typeable by both assays (kappa coefficient (kappa)=0.90). In addition, the precore codon 29 sequence identified by the INNO-LiPA HBV precore was confirmed by nucleotide sequencing in all seven samples analysed. However, the INNO-LiPA HBV precore identified precore promoter sequences much less efficiently. The prototype assay could identify codon 28/29 sequences from as little as 10 HBV genome equivalents in 10 microl serum, and in experiments using artificially prepared mixtures of variants could identify a minor component constituting 2.5% of the total viral DNA population. The INNO-LiPA HBV precore was also straightforward technically and rapid, and is therefore likely to be useful for epidemiological investigations into the prevalence, distribution and clinical significance of HBV precore variants.

DNA Probes↗
PubMed2003

Identification and distribution of different mRNA variants produced by differential splicing in the human phosphodiesterase 9A gene.

The transcript population of the human gene coding for a cGMP-dependent phosphodiesterase (PDE9A) has a complex structure. There is a high level of mRNA in intestinal and prostate tissues, a low level in blood, and intermediate in other tissues. More than 20 different variants produced by differential splicing have been observed and new exons have been identified both by PCR amplification and by the analysis of available EST sequences. In all cases the transcriptional start site is the same and no differential splicing is found in the exons coding for the catalytic domain of the protein. In some cases the protein produced by splice variants is truncated. The distribution of the splice variants is not homogeneous among the different tissues studied. The human, but not the mouse, PDE9A gene appears to have a complex regulation of expression by different isoforms.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2003

Identification of a new SERCA2 splice variant regulated during monocytic differentiation.

Sarco/endoplasmic reticulum-type calcium transport ATPases (SERCA enzymes) pump calcium ions from the cytosol into the endoplasmic reticulum. We report that in addition to the ubiquitously expressed SERCA2b isoform, a new splice variant of SERCA2 can be detected (SERCA2c) that arises from the inclusion of a short intronic sequence located between exons 20 and 21 of the SERCA2a isoform. Sequence analysis revealed classical splice donor and acceptor sites, as well as a branch-point site. Due to the presence in the new exon of an in-frame stop codon that is preceded by a 17 bp coding sequence, this mRNA potentially codes for a protein with a truncated C-terminus containing a short unique C-terminal peptide stretch. SERCA2c message was detected in epithelial, mesenchymal, and hematopoietic cell lines, as well as in primary human monocytes. Moreover, we found that during monocytic differentiation total SERCA2 ATPase expression is induced on the protein and mRNA level and that the novel SERCA2c messenger is also up-regulated during this process. These data indicate that the alternative splicing pattern of the 3(') region of the SERCA2 primary transcript is more complex than that previously thought and that this enzyme may be involved in the process of monocyte differentiation.

Alternative Splicing↗
PubMed2000

Identification and characterization of a truncated variant of the 5-hydroxytryptamine(2A) receptor produced by alternative splicing.

We have identified an alternatively spliced 5-hydroxytryptamine 2A receptor (5-HT(2A)-R) transcript by PCR of human brain cDNA using degenerate oligonucleotide primers to transmembrane (TM) domains 3 and 7 of the 5-HT(2)-R subfamily. The variant contains a 118-bp insertion at the exon II/III boundary of the 5-HT(2A)-R, which produces a frame shift in the coding sequence and a premature stop codon. PCR analysis showed that the truncated receptor (5-HT(2A-tr)) and native 5-HT(2A)-R were co-expressed in most brain tissues, with the highest levels being found in hippocampus, corpus collosum, amygdala and caudate nucleus. Western blot analysis of HEK-293 cells transfected transiently with a 5-HT(2A-tr) construct showed that a 30-kDa protein was expressed on cell membranes. Co-transfection studies showed no effect of the 5-HT(2A-tr) variant on 3H-ketanserin binding to the native 5-HT(2A)-R or on functional coupling of the 5-HT(2A)-R to 5-HT-stimulated Ca(2+) mobilization. The functional significance of the 5-HT(2A-tr) variant and other truncated receptors remains to be established.

Amino Acid Sequence↗