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PubMed1998

Identification and characterisation of a novel splice variant of synaptojanin1.

Synaptojanin1, the major constitutively active PtdInsP3 5-phosphatase activity in rat brain, is one of two closely related proteins both extensively spliced in their C-terminal proline rich domain. We describe here the discovery of a novel splice variant of synaptojanin1 which misses the major N-terminal part of the SAC1 domain. This deltaSAC-synaptojanin1 is expressed in rat brain tissue as shown by Northern and Western analysis. However, the deletion of the SAC1 domain does not alter PtdInsP3 5-phosphatase activity demonstrating that the SAC1 domain is not necessary for catalytic function.

Alternative Splicing↗

PubMed1999

Identification and characterization of a novel splice variant of MuSK.

MuSK is a receptor tyrosine kinase that initiates the formation of neuromuscular junctions in response to agrin. Little is known about the ligand-induced activation and kinase-dependent signalling that leads to the clustering of acetylcholine receptors. The ectodomain of these molecule is composed of four Ig-like domains. We describe here the isolation of a novel MuSK splice variant that lacks the third Ig-like domain in its ectodomain. The corresponding RNA is the result of alternative splicing which eliminates two exons. There is 10 times less mRNA for this shorter form than for the long form of MuSK and both forms are regulated coordinately. They decrease strongly after birth and are elevated in denervated muscle. Gene transfer by muscle injection of MuSK DNA into individual muscle fibers demonstrates that kinase-induced acetylcholine receptor clustering caused by overexpression of the two kinases does not depend on the presence of the third Ig-like domain.

Alternative Splicing↗

PubMed2001

Identification of a prostaglandin E2 receptor splice variant and its expression in rat tissues.

The intercellular signalling actions of the lipid mediators, the eicosanoids, are transduced by a family of seven transmembrane domain receptors. Members of this receptor family with high affinity for PGE2 are termed EP receptors. There are four known EP receptor genes that are transcribed to generate EP1, EP2, EP3 and EP4 receptors. Two of these receptor transcripts, EP1 and EP3, are further modified by RNA splicing to give multiple receptor isoforms. The EP3 receptor is known to have multiple splice variants in human (9 variants), cow (4 variants), mouse (3 variants) and rat (3 variants). In the rat the three EP3 splice variants differ in the sequence of the intracellular C-terminus. We have identified a fourth splice variant of the rat prostaglandin EP3 receptor that has a greatly truncated intracellular C-terminus when compared to the other EP3 receptor isoforms. Using nested RT-PCR we have shown that this novel splice variant is strongly expressed in rat brain and is also found in spinal cord, kidney and spleen.

Alternative Splicing↗

PubMed1998

Chromatographic and immunological identification of GnRH (gonadotropin-releasing hormone) variants. Occurrence of mammalian and a salmon-like GnRH in the forebrain of an eutherian mammal: Hydrochaeris hydrochaeris (Mammalia, Rodentia).

The molecular variants of Gonadotropin releasing hormone (GnRH) in brain extracts of the eutherian mammal Hydrochaeris hydrochaeris (Mammalia, Rodentia) were characterized. An indirect method combining reverse-phase high-performance liquid chromatography (RP-HPLC) and radioimmunoassay (RIA) with different antisera was used. Two different forebrain regions (olfactory bulbs and preoptic-hypothalamic region) were analyzed. Characterization of RP-HPLC fractions from preoptic-hypothalamic extracts with three different RIA systems revealed two immunoreactive GnRH (ir-GnRH) peaks coeluting with mammalian GnRH (mGnRH) and salmon GnRH (sGnRH) synthetic standards. These results were additionally supported by serial dilution studies with specific antisera. Similar results were obtained from olfactory bulb extracts with the same methodology. However, a third ir-GnRH peak in a similar position to that of chicken GnRH II (cIIGnRH) synthetic standard was revealed. As far as we know, this is the first report showing chromatographic and immunological evidences for the presence of a second GnRH variant in the forebrain of an eutherian mammal.

Animals↗

PubMed1998

Identification and characterization of a new splicing variant of vascular endothelial growth factor: VEGF183.

We report the discovery of a new splicing variant of vascular endothelial growth factor named VEGF183. It is six amino acids shorter than its closest relative, VEGF189, due to the utilization of a conserved alternate splicing donor site within exon 6a. Highly expressed in heart tissue, VEGF183 is detected in transiently transfected COS cells as 28-32-kDa monomers under reduced condition, and 46-kDa dimers under non-reduced condition - the functional unit for all VEGF isoforms.

Alternative Splicing↗

PubMed2002

Identification and characterization of a novel splice variant of the metabotropic glutamate receptor 5 gene in human hippocampus and cerebellum.

The G-protein coupled metabotropic glutamate receptor mGlu5 plays a pivotal role as a modulator of synaptic plasticity, ion channel activity and excitotoxicity. Two splice variants, hmGlu5a and -5b have been reported previously. During screening of a human brain cDNA library for hmGlu5a, we identified a novel variant (hmGlu5d) generated by alternative splicing at the C-terminal domain. The predicted hmGlu5d protein has a C-terminal 267 amino acid shorter than that of hmGlu5a. The pattern of mRNA expression of mGluR5 variants in human brain were analyzed by RT-PCR and in situ hybridization histochemistry. RT-PCR analysis demonstrated the presence of the hmGlu5d transcript, although at low level, in human whole brain, cerebellum, cerebral cortex and hippocampus. [3H]Quisqualate displayed similar affinity at the hmGlu5 splice variants (K(D) values of 80+/-8 and 54+/-17 nM for hmGlu5a and -5d receptors, respectively). For the five mGlu agonists studied, a similar rank order of potency was observed on both hmGlu5a and -5d receptors: quisqualate>glutamate>DHPG>L-CCGI approximately ACPD. MPEP inhibited the glutamate (2 microM)-induced [Ca(2+)](i) response in hmGlu5a and -5d-HEK293 cells also with similar potency (IC(50) values 25+/-1.5 and 20+/-1.4 nM, respectively). Therefore, the large truncation of the C-terminal tail of mGlu5 does not have any apparent major effect on the potency and efficacy of agonists as measured by the [Ca(2+)](i) responses or by activation of recombinant G-protein coupled inwardly rectifying K(+) (GIRK) channel currents. The only major functional difference is the increased sensitivity of hmGlu5d to protein kinase C (PKC)-mediated desensitization, relative to hmGlu5a.

Aged↗

PubMed1999

Identification of a human 5-HT6 receptor variant produced by alternative splicing.

The complexity of the 5-hydroxytryptamine (5-HT) (serotonin) receptor family has been increased by the findings that isoforms or splice variants exist for subtypes such as the 5-HT2B, 5-HT2C, 5-HT4 and 5-HT7 subtypes. Further molecular biological studies in our laboratory have demonstrated that a splice variant of the 5-HT6 receptor exists in the human brain. Experiments performed using a degenerate PCR approach from human caudate cDNA revealed a 5-HT6 receptor clone with a 289 bp deletion of the region coding for transmembrane IV through the third intracellular loop. This deletion produces a frameshift creating a downstream stop codon which results in a truncated protein containing 10 unique amino acids at its carboxyl end. The variant transcript occurs as a result of alternative splicing using an upstream donor site and the acceptor site from the first intron in the 5-HT6 receptor gene. The splicing pattern seen for this transcript was not detected in rat or mouse whole brain cDNA by PCR due to the lack of a consensus 5' donor site. Coexpression of the variant 5-HT6 transcript and the full length 5-HT6 transcript was observed in caudate and substantia nigra but not in hippocampus, cortex, cerebellum and thalamus. Transient transfection of a 5-HT6 variant construct into Cos-7 cells demonstrated that a truncated receptor was translocated to the membrane but appeared nonfunctional.

Alternative Splicing↗

PubMed1996

Identification and cloning of site C splice variants of plasma membrane Ca-ATPase in the gerbil cochlea.

Plasma membrane Ca-ATPase (PMCA) gene products were identified in the gerbil cochlea by reverse-transcription polymerase chain reaction (RT-PCR). Cochlear cDNA was amplified using PMCA isoform-specific primers from splice site C, the calmodulin binding domain. PCR products were cloned and sequenced. The putative housekeeping PMCA genes, 1b and 4b, as expected, were present in the gerbil cochlea and shared 98.6 and 100% amino acid homology with published rat sequences, at splice site C, respectively. PMCA2b, 3a and 3b splice variants also were detected in cochlear cDNAs and shared 95, 94.3 and 98% amino acid homology with their rat counterparts. PMCA isoforms 2 and 3 have been shown to occur in highly specialized tissues, such as muscle and brain, that require finely tuned regulation of intracellular free Ca2+ levels. The presence of several isoforms and splice variants of PMCA in the cochlea most probably reflects their differential expression among the several cell types that have been shown to contain immunoreactive PMCA. This suggests that the cochlea has developed complex mechanisms that finely tune intracellular Ca2+ levels in different highly specialized cell types.

Amino Acid Sequence↗

PubMed1999

Identification of an HLA-A11 serological variant and its characterization by sequencing based typing.

We have identified an HLA-A11 variant allele, A*1105, segregating in a Caucasoid family. The variant antigen expressed by this allele failed to cross-react with most Caucasoid anti-HLA-A11 antisera tested. Sequencing based typing has been used to characterize this new allele and this showed that it has a novel mutation at a polymorphic position (502) in exon 3. In comparison with A*1101, the mutation (A-->G) results in an amino acid change from positively-charged lysine to negative glutamate and this may explain the altered HLA-A11 serological profile exhibited by this antigen. The new allele was found in a patient with acute lymphoid leukaemia (ALL), her father and two siblings.

Alleles↗

PubMed2000

Identification of an HLA-B7 serological variant and its characterization by sequencing based typing.

We have identified an HLA-B*07 variant allele, B*0716, in a Caucasoid cadaver kidney donor. The HLA class I type by polymerase chain reaction using sequence-specific primers (PCR-SSP) was A*01, 32; B*07, 08; Cw*07. Serological typing, using monoclonal and polyclonal anti-HLA antisera, gave disparate results for the B antigens. Monoclonal antibodies identified B7 and B8 antigens but polyclonal antisera recognised only the B8 antigen. PCR using sequencing based typing (PCR-SBT) confirmed the presence of both B*0703 and B*0801 alleles but with a mutation in one of the alleles. The HLA-B*07 allele was isolated by allele-specific PCR and was shown to have a mutation, G-->T, at 292 in exon 2. This mutation changes codon 74, which encodes aspartic acid (GAC) present in all previously identified B*07 alleles, to tyrosine (TAC) in the variant. The serological results suggest that codon 74 is a crucial part of a B7 antigen-specific epitope recognised by tissue typing polyclonal antisera.

Alleles↗

PubMed2000

Identification of a new DRB302 allelic variant (DRB30209) by high-resolution sequence-based typing.

The HLA-DRB3/B4/B5 sequence-based typing method developed in this study in combination with PCR-SSP, enabled us to identify a new DRB3*02 allele, that was named as DRB3*0209 (GenBank accession number AF148518). This name has been officially assigned by the WHO Nomenclature Committee in May 1999. The new allele differs from DRB3*0207 by one substitution in codon 51 from AGG to ACG and another in codon 60 from TAC to TCC, resulting in aminoacid changes from Arg-->Thr (codon 51) and from Tyr-->Ser (codon 60). The DRB3*0209 allele was discovered in two related North Italian families. The fact that it was present in an hemizygous situation in three members of the paternal family and in one member of the secondary related family enabled us to isolate and sequence the new DRB3 allele without cloning, to identify its association with the DRB1 locus, and to generate an Epstein-Barr virus (EBV)-transformed cell line, now present in our ECBR (European Collection for Biomedical Research) Cell Line Bank.

Alleles↗

PubMed2001

Identification and sequence characterization of a novel HLA-A11 serological variant (HLA-A*1108).

In this report we describe the identification of a novel HLA-A*11 allele, HLA-A*1108, found in two individuals of a Spanish family. This new allele was detected during routine HLA typing by an atypical serological reactivity pattern and by inconclusive patterns obtained in DNA-based typing methods. The nucleotide sequence of exons 2 and 3 of HLA-A*1108 was identical to HLA-A*11011 except for two nucleotide substitutions at codons 152 (GCG-->GAG) and 156 (CAG-->CGG). These mutations change the non-charged amino acid alanine, at codon 152, to negative glutamic acid, and also non-charged glutamine, at codon 156, to positive arginine, which may explain its anomalous serological reactivity.

Alleles↗

PubMed2001

Identification of two new HLA-B22 variants, HLA-B5509 and B5606.

In our recent study using high-resolution HLA-B locus typing by sequence-based typing (SBT) we identified 9 new alleles in a total of 355 unrelated individuals (4). Three of them concerned an allele belonging to the B22 group. One of them, B*5607, showed the unusual presence of a Bw4 sequence motif, as described previously (5). In this report the other two B22 variants are described; one belonging to the B55 specificity and named B*5509; the other one being a B*56 allele and assigned B*5606, which brings the total number of alleles belonging to the B22 group to 18.

Alleles↗

PubMed1999

Human P2Y2 receptor polymorphism: identification and pharmacological characterization of two allelic variants.

1. In the process of cloning the human P2Y2 receptor in order to establish 1321N1 cell lines expressing this receptor, we detected a gene polymorphism characterized by an arginine 334 to cysteine 334 transition. 2. The frequency distribution of the polymorphism was studied in a European population. We observed that 66% of the tested persons are homozygotes R/R, 29% are heterozygotes R/C and 5% are homozygotes C/C. The frequency of the R allele was 0.8 versus 0.2 for the C allele. 3. We stably expressed each form of the human P2Y2 receptor into 1321N1 cells and isolated clones by limiting dilution. The effects of nucleotides and antagonists on inositol trisphosphate accumulation and cyclic AMP formation were compared between the two cell lines. 4. The time-courses of inositol trisphosphate accumulation as well as concentration-response curves characterizing the effects of UTP, ATP, AP4A and ATP gamma S were mostly similar, except for slight kinetic differences (slower time-course with the 334C form). 5. The sensitivity to pertussis toxin of inositol trisphosphates accumulation was critically dependent on the agonist concentration and stimulation duration, suggesting the involvement of a Gi.0 protein during the early stimulation by low nucleotide concentrations. No inhibition of cyclic AMP accumulation could be detected. These properties were observed with both polymorphic receptors.

Adenylyl Cyclases↗

PubMed2000

Genomic structure, chromosomal localization and identification of mutations in the xeroderma pigmentosum variant (XPV) gene.

The xeroderma pigmentosum variant (XP-V) is one of the most common forms of this cancer-prone syndrome. XP groups A through G are characterized by defective nucleotide excision repair, whereas the XP-V phenotype is proficient in this pathway. The XPV gene encodes DNA polymerase eta, which catalyzes an accurate translesion synthesis, indicating that the XPV gene contributes tumor suppression in normal individuals. Here we describe the genomic structure and chromosomal localization of the XPV gene, which includes 11 exons covering the entire coding sequence, lacks a TATA sequence in the upstream region of the transcription-initiation, and is located at the chromosome band 6p21.1-6p12. Analyses of patient-derived XP-V cell lines strongly suggested that three of four cell lines carried homozygous mutations in the XPV gene. The fourth cell line, XP1RO, carried heterozygous point mutations in the XPV gene, one of which was located at the splice acceptor site of exon 2, resulting in the omission of exon 2 from the mature mRNA. These findings provide a basis for diagnosis and therapy of XP-V patients.

Alleles↗

PubMed1981

Identification of "buried" lysine residues in two variants of chloramphenicol acetyltransferase specified by R-factors.

Two variants of chloramphenicol acetyltransferase which are specified by genes on plasmids found in Gram-negative bacteria were subjected to amidination with methyl acetimidate to determine the relative reactivity of surface lysine residues and to search for unreactive or "buried" amino groups which might contribute to stabilization of the native tetramers. Representative examples of the type-I and type-III variants of chloramphenicol acetyltransferase were found to have one lysine residue each in the native state which appears to be inaccessible to methyl acetimidate. The uniquely unreactive residue of the type-I protein is lysine-136, whereas the lysine that is "buried" in the type-III enzyme is provisonally assigned to residue 38 of the prototype sequence. It is suggested that the lysine residue in each case participates in the formation of an ion pair at the intersubunit interface and that the two amino groups in question occupy functionally equivalent positions in the quaternary structures of their respective enzyme variants. Lysine-136 of type-I enzyme is also uniquely unavailable for modification by citraconic anhydride, a reagent used to disrupt the quaternary structure of the native enzyme. Contrary to expectation, exhaustive citraconylation fails to dissociate the tetramer, but does destroy catalytic activity. Removal of citraconyl groups from modified chloramphenicol acetyltransferase is accompanied by a full region of catalytic activity. Analysis of the rate of hydrolysis of citraconyl groups from the modified tetramer by amidination of unblocked amino groups with methyl [14C]acetamidate reveals difference in lability for several of the ten modified lysine residues. Although the unique stability of the quaternary structure of chloramphenicol acetyltransferase may be due to strong hydrophobic interactions, it is argued that lysine-136 may contribute to stability via the formation of an ion pair at the subunit interface.

Acetyltransferases↗

PubMed2005

Variant tricarboxylic acid cycle in Mycobacterium tuberculosis: identification of alpha-ketoglutarate decarboxylase.

Mycobacterium tuberculosis (Mtb) has adapted its metabolism for persistence in the human macrophage. The adaptations are likely to involve Mtb's core intermediary metabolism, whose enzymes have been little studied. The tricarboxylic acid cycle is expected to yield precursors for energy, lipids, amino acids, and heme. The genome sequence of Mtb H37Rv predicts the presence of a complete tricarboxylic acid cycle, but we recently found that alpha-ketoglutarate dehydrogenase (KDH) activity is lacking in Mtb lysates. Here we showed that citrate synthase, aconitase, isocitrate dehydrogenase, fumarase, malate dehydrogenase, and succinate dehydrogenase, but not KDH, are present, raising the possibility of separate oxidative and reductive half-cycles. As a potential link between the half-cycles, we found that Rv1248c, annotated as encoding SucA, the putative E1 component of KDH, instead encodes alpha-ketoglutarate decarboxylase (Kgd) and produces succinic semialdehyde. Succinic semialdehyde dehydrogenase activity was detected in Mtb lysates and recapitulated with recombinant proteins GabD1 (encoded by Rv0234c) and GabD2 (encoded by Rv1731). Kgd and GabD1 or GabD2 form an alternative pathway from alpha-ketoglutarate to succinate. Rv1248c, which is essential or required for normal growth of Mtb [Sassetti, C., Boyd, D. H. & Rubin, E. J. (2003) Mol. Microbiol 48, 77-84] is the first gene shown to encode a Kgd. Kgd is lacking in humans and may represent a potential target for chemotherapy of tuberculosis.

Bacterial Proteins↗

PubMed1996

Identification of a novel cysteine string protein variant and expression of cysteine string proteins in non-neuronal cells.

Cysteine string proteins (Csps) are synaptic vesicle proteins thought to be involved in calcium-dependent neurotransmitter release at nerve endings. Here, we report the cloning of two Csp variants, termed Csp1 and Csp2, from bovine adrenal medullary chromaffin cells. The bovine Csp1 appears to be the homologue of rat brain Csp, sharing 95% identity at the amino acid level. The nucleotide sequence of csp2 is identical with that of csp1 except for a 72-base insert which introduces a stop codon into the coding sequence, which would be predicted to result in a truncated protein 3.3 kDa smaller than Csp1. Furthermore, polymerase chain reaction analysis detected homologues of Csp1 and Csp2 in rat kidney, liver, pancreas, spleen, lung, and adrenal gland. Expression of Csps in non-neuronal tissues was confirmed by Northern blotting and by immunoblotting with anti-Csp1 antiserum which also demonstrated expression of both full-length and truncated Csps in spleen. The widespread tissue distribution is inconsistent with a role of Csps as specific regulators of presynaptic calcium channels as previously proposed. We suggest that Csps may have a more general role in membrane traffic in non-neuronal as well as neuronal cells.

Adrenal Medulla↗