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PubMed2001

Identification of an alternatively spliced variant of human CD86 mRNA.

CD86 is a costimulatory molecule constitutively expressed by human antigen presenting cells which interacts with CD28 and CTLA-4 expressed by T cells. We have recently reported the identification of an alternatively spliced CD86 mRNA variant (CD86deltaTM) characterized by the deletion of exon 6 which encodes for the transmembrane domain. We report here the identification of an alternatively spliced variant (called CD86deltaEC) expressed by nonstimulated human monocytes and characterized by the deletion of exons 4 and 5 which encode for the extracellular V-like and C-like domains, respectively. The activation of monocytes by IFNgamma (i) induces the preferential expression of the transcript encoding for the membrane form and (ii) increases the expression of CD86 and of the accessory molecules CD40, CD49d and CD54. These results suggest that resting human monocytes may constitutively express different forms of CD86 which can then influence the activation of T cells.

Alternative Splicing↗

PubMed1993

The molecular weights of twelve apolipoprotein(a) variants, determined using haptoglobin 2-2 polymer molecular weight standards.

Apolipoprotein(a) [apo(a)] variants were characterized in 398 sera by immunoblotting: (a) by molecular weight, using a haptoglobin 2-2 polymeric series as standards, and (b) by nomenclature, using serum pools containing previously characterized apo(a) variants as standards. The haptoglobin 2-2 standard curve (172-859 kDa) alleviates the necessity of obtaining molecular weights by extrapolation. Among the 398 sera, 40.2% had double apo(a) bands (54 phenotypes), 58.0% had a single apo(a) band and 1.8% were null (no bands observed). An inverse, though non-monotonic, relationship was observed between apolipoprotein(a) molecular weight and serum lipoprotein(a) [Lp(a)] concentration. Due to the large size of apo(a) and the relatively small increment between variants (15-16 kDa), molecular weight could not be used alone to characterize variants. Even with a CV of 3-4%, there was an overlap between variant molecular weight estimates. However, in combination with the identification of variants by comparison with standards, the haptoglobin 2-2 standard curve could be used to obtain mean molecular weight estimates for each variant. 12 distinct variants were identified among the sera, with apparent mean molecular weights of 314, 388, 410, 433, 454, 466, 503, 519, 528, 543, 553 and 572 kDa, respectively. These molecular weight estimates are consistent with the theoretical molecular weight range for apo(a) variants, calculated from sequence and carbohydrate analysis, of 238-643 kDa.

Apolipoproteins A↗

PubMed2002

Multiplex PCR strategy for rapid identification of structural types and variants of the mec element in methicillin-resistant Staphylococcus aureus.

Full characterization of methicillin-resistant Staphylococcus aureus (MRSA) requires definition of not only the bacterial genetic background but also the structure of the complex and heterologous mec element these bacteria carry, which is associated with drug resistance determinant mecA. We report the development, validation, and application of a multiplex PCR strategy that allows quick presumptive characterization of the mec element types based on the structural features that were shown to be typical of mec elements carried by several MRSA clones. The strategy was validated by using a representative collection of pandemic MRSA clones in which the full structure of the associated mec elements was previously determined by hybridization and PCR screenings and also by DNA sequencing. The method was tested together with multilocus sequence typing and other typing methods for the characterization of 18 isolates representative of the MRSA clones recovered during a hospital outbreak in Barcelona, Spain. The multiplex PCR was shown to be rapid, robust, and capable in a single assay of identifying five structural types of the mec element among these strains, three major and two minor variants, each one of which has been already been seen among MRSA characterized earlier. This technique should be a useful addition to the armamentarium of molecular typing tools for the characterization of MRSA clonal types and for the rapid tentative identification of structural variants of the mec element.

Bacterial Proteins↗

PubMed1994

Developmentally regulated expression of linker-histone variants in vertebrates.

The identification of histone H1 variants in vertebrates suggests that these proteins may have specialized functions. During embryonic development, a correspondence between the expression of each of the linker-histone variants and the proliferative and transcriptional activity of embryonic cells can be observed. Analysis of the developmentally regulated expression of these variants leads to the subdivision of these variants into distinct classes. This subdivision may also provide insight into the significance of the differential expression of variants and the roles individual linker histones have in chromatin structure and function.

Amino Acid Sequence↗

PubMed2005

Identification and characterization of splice variants of the human P2X7 ATP channel.

The P2X7 channel is a member of the P2X family of ligand-gated ion channels which respond to ATP as the endogenous agonist. Studies suggest that P2X7 has a potentially pivotal role in inflammatory responses largely stemming from its role in mediating the release of IL-1beta in response to ATP. We report the identification of seven variants of human P2X7 which result from alternative splicing. Two of these variants (one lacking the first transmembrane domain, the second lacking the entire cytoplasmic tail) were compared to the full-length channel. Real-time PCR analysis demonstrated that both variants were expressed in various tissues and that the cytoplasmic tail deleted variant is highly expressed. Deletion of the first transmembrane domain resulted in a non-functional channel. Deletion of the cytoplasmic tail did not affect ion movement but severely affected the ability to form a large pore and to induce activation of caspases.

Adenosine Triphosphate↗

PubMed1984

The fragile-X syndrome, IV. Progress towards the identification of linked restriction fragment length variants (RFLVs).

We present our strategy and progress towards the identification of rare restriction fragment length variants (RFLVs) segregating with the fragile-X syndrome. DNA from a carrier mother and two retarded sons was digested with 7 restriction endonucleases and Southern blots were probed with cloned unique X-chromosomal sequences. Two of 17 cloned segments tested revealed RFLVs between the X-chromosomes of the carrier mother. One of them detected variants using Pvu II and Msp I. The Pvu II and Msp I alleles found on the X-chromosome bearing the fragile-X mutation were not found in 31 and 22 random X-chromosomes, respectively. The other probe detected variants using Pvu II and Taq I. The Taq I allele present on the X-chromosome with the fragile-X mutation was found in 23 out of 25 random X-chromosomes, while the Pvu II allele was not found in 21 random X-chromosomes. One of these probes and two other cloned unique X-chromosome sequences were localized distal to Xq26 by in situ hybridization to prometaphase chromosomes and by probing Southern blots containing DNA from a deleted X-chromosome. These are being used for linkage analysis in an extended family.

Autoradiography↗

PubMed1996

Use of chimeric recombinant polypeptides to analyse conformational, surface epitopes on trypanosome variant surface glycoproteins.

Identification of surface-exposed epitopes on the variant surface glycoproteins (VSGs) of African trypanosomes has been complicated by the observation that most such epitopes are highly conformational. As a result, whenever the molecule is broken down for analysis, the epitope is generally lost. We have exploited the existence of closely related gene families to create chimeric molecules in which particular segments of one VSG are placed in the analogous position of a related but antigenically distinct VSG. The process is used in both a positive and negative manner, so that the epitope can be specifically added or destroyed in a given chimera. As an example, we have used this approach to identify the regions involved in reactivity to a monoclonal antibody specific for VSG117 on the surface of live trypanosomes. We show that while deletion of almost any region of VSG117 results in loss of reactivity to this monoclonal antibody, substituting particular regions with the corresponding segment of the structurally related but antigenically distinct VSG FM8.5 restores reactivity in most but not all cases, thereby delimiting the antigenically key regions. Likewise, substituting key regions from VSG117 into FM8.5 confers reactivity on the resulting chimeras. This approach circumvents some of the problems that result from the highly conformational nature of VSG and should allow further elucidation of the biologically relevant antigenic topology of VSGs.

Africa↗

PubMedDate not supplied

Detection and characterization of variant and modified structures of proteins in blood and tissues by mass spectrometry.

Some variant proteins cause diseases, and some diseases result in increases of proteins with abnormally modified structures. The detection, characterization, and estimation of the relative amounts of protein variants and abnormally modified proteins are important for clinical diagnosis and for elucidation of the mechanisms of the pathogenesis of diseases. Analysis of the covalent structures of proteins using matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS) and liquid chromatography-electrospray ionization MS (LC-ESI-MS), which had been developed by the early 1990s, have largely replaced analyses by conventional protein chemistry. Here, we review the detection and characterization of hemoglobin variants, HbA1c measurement, detection of carbohydrate-deficient transferrin, and identification of variants of transthyretin (TTR) and Cu/Zn-superoxide dismutase (SOD-1) using soft ionization MS. We also propose the diagnostic application of the signals of modified forms of TTR, that is, S-sulfonated TTR and S-homocysteinyl TTR. The relative peak height ratio of the abnormal/normal components gives valuable information about the instability of variants and enables the detection of unstable Hb subunits or thalassemia heterozygotes. We found unique modified structures of TTR that suggested changes in amyloid fibrils.

Amino Acid Sequence↗

PubMed1999

Alleic variants of human melatonin 1a receptor: function and prevalence in subjects with circadian rhythm sleep disorders.

The human melatonin 1a (hMella) receptor gene was screened for mutations using genomic DNA samples from patients with circadian rhythm sleep disorders and control subjects by single strand conformational polymorphism analysis (SSCP). We found seven mutations, two of which predict amino acid changes R54W and A157V, respectively. The prevalence of the R54W variant and that of the A157V variant were several times more common in non-24-h sleep-wake syndrome subjects than among control subjects, although the incidence was not significant in our study group. When expressed in COS-7 cells, the R54W mutant receptor exhibited significantly reduced B(max) and slightly enhanced affinity (reduced K(d)) compared to the wild type receptor, while the A157V variant receptor showed similar binding characteristics to the wild type. The identification of variants in the hMella receptor will provide a useful tool for analyzing genetic predisposition toward various diseases related to melatonin function and to clarify the physiological role of melatonin receptors in humans.

Alleles↗

PubMed2002

Ferrochelatase gene polymorphism analysis for accurate genetic counselling in erythropoietic protoporphyria.

It has recently been shown that most cases of clinically overt erythropoietic protoporphyria (EPP) result from coinheritance of a mutated ferrochelatase gene and a commonly occurring low-expression normal variant allele. The identification of two polymorphic variant sequences associated with this low-expression allele now enables improved predictive counselling for couples where one partner has EPP. We describe a patient and his spouse in whom we have used such genetic analysis to provide an accurate estimate of the chance that their future offspring may suffer from EPP.

Adult↗

PubMed2005

Polygenic inherited predisposition to breast cancer.

The known breast cancer predisposing genes account for only about 20% of inherited susceptibility. Epidemiological analyses suggest that much of the remaining 80% is explained by the combined effect of many individually weak genetic variants, rather than by further rare, highly penetrant mutations. In the near term, identification of variants may indicate new pathways or mechanisms in breast cancer development. The polygenic model implies a wide distribution of risk in the population. In the longer term, it may be possible to construct individual risk profiles to guide public health interventions. The search for genetic variants has so far proved difficult. A key unanswered question is the "genetic architecture" of predisposition-that is, strong or weak alleles, common or rare. We describe a genome-wide scan designed to provide a first-pass answer to this question.

Alleles↗

PubMed1995

Applications of minisatellite variant repeat (MVR) mapping for maternal identification from remains of an infant and placenta.

Minisatellite variant repeat (MVR) mapping using the polymerase chain reaction (PCR) at D1S8 (MS32) was applied to a practical forensic case of an infant and placenta found in an incinerator. They were thought to be left for a few days postmortem, and the infant was severely burnt when found. DNA was extracted from the infantile muscle and maternal placental hematoma. MVR-PCR analysis as well as other common DNA typing (D1S80, HLA-DQA1) were performed on both DNA samples. Both MVR diploid codes were matched although some extra faint bands in the ladder were observed from the maternal placental sample, which probably indicated superimposing of an allele derived only from the mother, and not the infant. In order to detect the original maternal alleles, three flanking polymorphic sites were typed and allele-specific MVR-PCR was performed. Finally, one maternal allele not inherited by the infant and two alleles from the infant were typed. Two alleles suggested the infant and/or mother was Japanese. The two diploid codes including one possibly from the mother were deduced and compared with other codes in the databases for evaluating the discriminating power.

Base Sequence↗

PubMed2005

Isolation of a hop-sensitive variant of Lactobacillus lindneri and identification of genetic markers for beer spoilage ability of lactic acid bacteria.

We have isolated a hop-sensitive variant of the beer spoilage bacterium Lactobacillus lindneri DSM 20692. The variant lost a plasmid carrying two contiguous open reading frames (ORF s) designated horB(L) and horC(L) that encode a putative regulator and multidrug transporter presumably belonging to the resistance-nodulation-cell division superfamily. The loss of hop resistance ability occurred with the loss of resistance to other drugs, including ethidium bromide, novobiocin, and cetyltrimethylammonium bromide. PCR and Southern blot analysis using 51 beer spoilage strains of various species of lactic acid bacteria (LAB) revealed that 49 strains possessed homologs of horB and horC. No false-positive results have been observed for nonspoilage LAB or frequently encountered brewery isolates. These features are superior to those of horA and ORF 5, previously reported genetic markers for determining the beer spoilage ability of LAB. It was further shown that the combined use of horB/horC and horA is able to detect all 51 beer spoilage strains examined in this study. Furthermore sequence comparison of horB and horC homologs identified in four different beer spoilage species indicates these homologs are 96.6 to 99.5% identical, which is not typical of distinct species. The wide and exclusive distribution of horB and horC homologs among beer spoilage LAB and their sequence identities suggest that the hop resistance ability of beer spoilage LAB has been acquired through horizontal gene transfer. These insights provide a foundation for applying trans-species genetic markers to differentiating beer spoilage LAB including previously unencountered species.

Anti-Bacterial Agents↗

PubMed1995

Identification of polymorphisms and sequence variants in the human homologue of the mouse natural resistance-associated macrophage protein gene.

The most common mycobacterial disease in humans is tuberculosis, and there is evidence for genetic factors in susceptibility to tuberculosis. In the mouse, the Bcg gene controls macrophage priming for activation and is a major gene for susceptibility to infection with mycobacteria. A candidate gene for Bcg was identified by positional cloning and was designated "natural resistance-associated macrophage protein gene" (Nramp1), and the human homologue (NRAMP1) has recently been cloned. Here we report on (1) the physical mapping of NRAMP1 close to VIL in chromosome region 2q35 by PCR analysis of somatic cell hybrids and YAC cloning and (2) the identification of nine sequence variants in NRAMP1. Of the four variants in the coding region, there were two missense mutations and two silent substitutions. The missense mutations were a conservative alanine-to-valine substitution at codon 318 in exon 9 and an aspartic acid-to-asparagine substitution at codon 543 in the predicted cytoplasmic tail of the NRAMP1 protein. A microsatellite was located in the immediate 5' region of the gene, three variants were in introns, and one variant was located in the 3' UTR. The allele frequencies of each of the nine variants were determined in DNA samples of 60 Caucasians and 20 Asians. In addition, we have physically linked two highly polymorphic microsatellite markers, D2S104 and D2S173, to NRAMP1 on a 1.5-Mb YAC contig. These molecular markers will be useful to assess the role of NRAMP1 is susceptibility to tuberculosis and other macrophage-mediated diseases.

Alleles↗

PubMed1980

Quantitation of light chain synthesis in myeloma x spleen cell hybrids and identification of myeloma chain loss variants using radioimmunoassay.

A radioimmunoassay specific for the MOPC 21 kappa (K) myeloma chain of NSI and X63 myeloma x spleen cell hybrids was used to study light chain secretion in myeloma-hybrid lines. The M1 series of rat spleen cell x NSI mouse myeloma hybrid lines was chosen to illustrate the application of the radioimmunoassay for K chain quantitation and identification of K chain loss variants. Most of these lines secrete H (specific heavy), L (specific light), and K (myeloma kappa) chains, i.e., are HLK lines. Assays specific for rat L chain and mouse K chain showed that the ratio of L/K chain secreted by 6 different hybrid HLK lines ranged from 1.1 to 12.4. Using the rapid radioimmunoassay screening procedure, HL clonal variants which had lost K chain secretion were isolated at a frequency of approximately 10(-2) and characterized. K chain loss was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of radiolabelled secreted products. Stability of one HL line and its HLK parent was examined during 9 months of growth in vitro. The HL line remained stable, while antibody secreted by the HLK line became inactive, apparently due to overgrowth by clonally dominant HK cells which no longer secreted specific L chains. The radioimmunoassay appears to detect MOPC 21 kappa chain variable region determinants. Therefore, although used here with rat-mouse hybrids, it should also be possible to use the assay to obtain mouse-mouse variant hybrid lines secreting antibody of improved homogeneity.

Animals↗

PubMed1988

Differential sensitivity to natural cell-mediated cytotoxicity of two rat colon adenocarcinoma variants differing in their tumorigenicity: identification of the effector cells as natural killer cells.

DHD/K12 TRb (PROb) and DHD/K12 TSb (REGb) are two cancer cell variants originating from the same rat colon adenocarcinoma. They differ in their tumorigenicity: when inoculated into syngeneic BDIX rats, PROb cells induce progressive tumors whereas REGb cells induce tumors which always regress. As previously described, there is an inverse relation between their tumorigenicity and their susceptibility to NCMC mediated by syngeneic spleen or peripheral blood lymphocytes: PROb cells are significantly less sensitive to NCMC than REGb cells. This suggests a role for NCMC in the regression of REGb tumors. In this work the BDIX NCMC effector cells active in vitro against REGb cells were identified as NK cells according to four criteria: (1) efficacy in a 4-h 51Cr release assay, (2) sensitivity to anti-asGM1 antibody plus complement, (3) LGL morphology, and (4) ability to bind with the same affinity REGb and YAC-1 cells. In spleen, these NK cells were heterogeneous with respect to their asGM1 surface density and their morphology. PROb cells were not lysed by these NK cells in a short-term cytotoxicity assay, but only in a 16-h assay. It was shown that PROb and REGb cells were bound with the same affinity by NK cells, thus they certainly differ in their ability to resist to NK lytic mechanisms. This difference could play a role in the different tumorigenicity of the two variants.

Adenocarcinoma↗

PubMed2007

Genotype and childhood sexual trauma moderate neurocognitive performance: a possible role for brain-derived neurotrophic factor and apolipoprotein E variants.

BACKGROUND: Limited success in the identification of genetic variants underpinning psychiatric illness has prompted attempts to elucidate gene-environment interactions and illness-associated endophenotypes. Here we measured childhood sexual abuse, a potential environmental risk factor, and verbal and visual recall and recognition memory, a possible illness-associated endophenotype in a cohort of bipolar disorder (BPD) subjects and their relatives. We predicted that memory would be affected by sexual trauma and that a number of functional polymorphisms previously implicated in BPD and cognition would moderate the effect of psychological trauma on memory. METHODS: A cohort of 350 individuals from 47 BPD families was recruited, tested with a neuropsychological battery, and given the Childhood Trauma Questionnaire (CTQ). Eleven different genetic variants previously found to be relevant to BPD or memory dysfunction were typed. RESULTS: As predicted, scores on the sexual abuse scale of the CTQ were negatively associated with memory performance. Furthermore, the low-activity Met allele of the brain-derived neurotrophic factor (BDNF) gene and the epsilon4 allele of the apolipoprotein E gene interacted with sexual abuse scores to result in reduced memory test performance. CONCLUSIONS: Apolipoprotein E and BDNF exert a neurotrophic effect in response to cellular injury. Their possible moderation of the association between sexual abuse and memory performance might indicate that there is some degree of overlap in the pathophysiological mechanisms by which psychological and physical trauma impact brain function. The finding of an environmental effect on memory performance and a gene-environment interaction on this hypothetical endophenotype of BPD illustrates the difficulty of identifying genetically and phenotypically simple intermediate traits for molecular genetic studies.

Adolescent↗

PubMed2005

Identification of a novel splice variant of X-linked inhibitor of apoptosis-associated factor 1.

XAF1 (XIAP-associated factor 1) binds to XIAP and blocks its anti-apoptotic activity. It has been reported that XAF1 is mainly expressed in normal tissues but is missing or present at low levels in most cancer cell lines, which implies a tumor-suppressing function. In the present study we describe the identification of a novel splice variant of human XAF1, designated XAF1C, which contains a cryptic exon. Incorporation of this exon (exon 4b) into the mRNA introduces an in-frame stop codon, resulting in a shortened open-reading frame (ORF) of 495 nucleotides. This ORF is predicted to encode a 164 amino acid (AA) protein lacking the C-terminal domain of the previously described XAF1(A), but containing a unique 24 AA carboxy terminus. Like XAF1(A), XAF1C mRNA expression was detected in a variety of human cancer cell lines and also in normal human tissues. The ratio of XAF1(A) and XAF1C mRNA expression differs amongst the cell lines tested, suggesting differential mRNA stabilities and/or the existence of tissue- or cell type-specific splicing regulation. In transfected cells, xaf1c encodes a truncated protein of 18kDa, which is distributed primarily in the nucleus.

Adaptor Proteins, Signal Transducing↗