Identification of a novel polymorphism (IVS6-33C->G) and two novel rare variants (IVS6-42delT and IVS6-43delA) in RPE65 gene.
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Carrier Proteins↗
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Identification of a 36-kDa fibronectin-binding protein expressed by a virulent variant of Leptospira interrogans serovar icterohaemorrhagiae.
We investigated the ability of a virulent strain of Leptospira interrogans serovar icterohaemorrhagiae, its isogenic avirulent variant and a saprophytic strain to bind fibronectin using alkaline phosphatase-labelled fibronectin. A single 36-kDa fibronectin-binding protein was expressed only by the virulent strain and was located in the outer sheath according to proteinase K treatment results. The interaction of this protein with fibronectin was specific and the region of fibronectin bound to this potential adhesin overlapped the gelatin-binding domain. The inability of a RGDS synthetic peptide to inhibit the binding of fibronectin indicated that the cell-binding domain was not involved in this interaction. Considering the wide distribution of fibronectin within a host and the diversity of mammals involved in the epidemiology of leptospirosis, its implication in the cell attachment process of virulent leptospires is coherent with the multiplicity of target cells.
Adhesins, Bacterial↗
Identification and characterization of cul-3b, a novel hominine CUL-3 transcript variant.
AIM: To identify genes related to the human testis development by substrate hybridization technique. METHODS: A human testis cDNA microarray was constructed and hybridized with probes prepared from human adult and fetal testes and spermatozoa mRNAs by reverse transcription reactions. The differentially expressed genes were sequenced. And a newly identified cullin-3 (CUL-3) transcript variant (designated cul-3b) was bio-informatically analyzed with an online GenBank database. Multi-tissue reverse transcription polymerase chain reaction (RT-PCR) was used to determine the tissue expression profile of cul-3b. RESULTS: Cul-3b, a novel CUL-3 transcript variant, was identified. The expression level of cul-3b in adult testes was 3.79-fold higher than that in fetal ones. Cul-3b differed from cul-3 (including NM_003590 and AY337761) in the opening reading frame and had three internal ribosomal entry sites IRESes in the 5'-UTR. These led to a 24 amino acid (aa) truncation at N-terminus of CUL-3b as compared with CUL-3 and a more motivated expression pattern of cul-3b under some strict circumstances. Additionally, cul-3b expressed ubiquitously in human tissues according to multi-tissue RT-PCR. CONCLUSION: Cul-3b is a novel transcript variant of CUL-3, which may be important not only for the development of human testis but also for that of other organs.
Base Sequence↗
Identification and functional analysis of common human flavin-containing monooxygenase 3 genetic variants.
Flavin-containing monooxygenases (FMOs) are important for the disposition of many therapeutics, environmental toxicants, and nutrients. FMO3, the major adult hepatic FMO enzyme, exhibits significant interindividual variation. Eighteen FMO3 single-nucleotide polymorphism (SNP) frequencies were determined in 202 Hispanics (Mexican descent), 201 African Americans, and 200 non-Latino whites. Using expressed recombinant enzyme with methimazole, trimethylamine, sulindac, and ethylenethiourea, the novel structural variants FMO3 E24D and K416N were shown to cause modest changes in catalytic efficiency, whereas a third novel variant, FMO3 N61K, was essentially devoid of activity. The latter variant was present at an allelic frequency of 5.2% in non-Latino whites and 3.5% in African Americans, but it was absent in Hispanics. Inferring haplotypes using PHASE, version 2.1, the greatest haplotype diversity was observed in African Americans followed by non-Latino whites and Hispanics. Haplotype 2A and 2B, consisting of a hypermorphic promoter SNP cluster (-2650C>G, -2543T>A, and -2177G>C) in linkage with synonymous structural variants was inferred at a frequency of 27% in the Hispanic population, but only 5% in non-Latino whites and African Americans. This same promoter SNP cluster in linkage with one or more hypomorphic structural variant also was inferred in multiple haplotypes at a total frequency of 5.6% in the African-American study group but less than 1% in the other two groups. The sum frequencies of the hypomorphic haplotypes H3 [15,167G>A (E158K)], H5B [-2650C>G, 15,167G>A (E158K), 21,375C>T (N285N), 21,443A>G (E308G)], and H6 [15,167G>A (E158K), 21,375C>T (N285N)] was 28% in Hispanics, 23% in non-Latino whites, and 24% in African Americans.
Black or African American↗
Identification of the major spliceosomal RNAs in Dictyostelium discoideum reveals developmentally regulated U2 variants and polyadenylated snRNAs.
Most eukaryotic mRNAs depend upon precise removal of introns by the spliceosome, a complex of RNAs and proteins. Splicing of pre-mRNA is known to take place in Dictyostelium discoideum, and we previously isolated the U2 spliceosomal RNA experimentally. In this study, we identified the remaining major spliceosomal RNAs in Dictyostelium by a bioinformatical approach. Expression was verified from 17 small nuclear RNA (snRNA) genes. All these genes are preceded by a putative noncoding RNA gene promoter. Immunoprecipitation showed that snRNAs U1, U2, U4, and U5, but not U6, carry the conserved trimethylated 5' cap structure. A number of divergent U2 species are expressed in Dictyostelium. These RNAs carry the U2 RNA hallmark sequence and structure motifs but have an additional predicted stem-loop structure at the 5' end. Surprisingly, and in contrast to the other spliceosomal RNAs in this study, the new U2 variants were enriched in the cytoplasm and were developmentally regulated. Furthermore, all of the snRNAs could also be detected as polyadenylated species, and polyadenylated U1 RNA was demonstrated to be located in the cytoplasm.
Animals↗
Gelsolin-related amyloidosis. Identification of the amyloid protein in Finnish hereditary amyloidosis as a fragment of variant gelsolin.
The Finnish type of familial amyloidosis is a systemic disease characterized by progressive cranial neuropathy, corneal lattice dystrophy, and distal sensimotor neuropathy. Amyloid fibrils were isolated from the kidney and heart of a patient with Finnish amyloidosis. After solubilization, the amyloid proteins were fractionated by gel filtration and purified by reverse-phase HPLC. Complete amino acid sequence analyses show that the two amyloid components obtained are fragments of gelsolin, an actin-modulating protein occurring in plasma and the cytoskeleton. The larger component represents residues 173-243 and the minor component residues 173-225, respectively, of mature gelsolin. When compared with the predicted primary structure of human gelsolin a single amino acid substitution is present in amyloid: at position 15 of the amyloid proteins an asparagine is found instead of an aspartic acid residue at the corresponding position (187) in gelsolin. Antibodies to a dodecapeptide of the amyloidogenic region of gelsolin specifically stain the tissue amyloid deposits in Finnish hereditary amyloidosis. The results show that the amyloid subunit protein in Finnish hereditary amyloidosis represents a new type of amyloid that is derived from an actin filament-binding region of a variant gelsolin molecule by limited proteolysis.
Amino Acid Sequence↗
Identification of Mutations Causing 6-Pyruvoyl- Tetrahydrobiopterin Synthase Deficiency in Polish Patients With Variant Hyperphenylalaninemia.
Background: 6-Pyruvoyl-tetrahydrobiopterin synthase (PTPS) is required for biosynthesis of tetrahydrobiopterin, the cofactor of various enzymes including the hepatic phenylalanine hydroxylase. Mutations in the PTS gene result in a variant type of hyperphenylalaninemia, requiring cofactor replacement therapy for treatment. Methods and Results: Four Polish patients with PTPS deficiency were screened for mutations in the PTS gene. Three novel mutations E35G, N36K, and F100V were identified. In one patient, a known mutation D136V was identified in both PTS alleles. Conclusions: Mutation D136V present in both alleles was proposed to be connected with a mild form of PTPS deficiency. The other three mutations were found in heterozygous patients with a central type of PTPS deficiency. D136V mutation is a common mutation in the Polish population.
Journal Article↗
Identification of a defect in the intracellular trafficking of a Kell blood group variant.
Blood group polymorphisms have been used as tools to study the architecture of the red blood cell (RBC) membrane. Some blood group variants have reduced antigen expression at the cell surface. Understanding the underlying mechanism for this reduced expression can potentially provide structural information and help to elucidate protein trafficking pathways of membrane proteins. The Kp(a+) phenotype is a variant in the Kell blood group system that is associated with a single amino acid substitution (R281W) in the Kell glycoprotein and serologically associated with a weakened expression of other Kell system antigens by an unknown mechanism. We found by immunoblotting of RBCs that the weakening of Kell antigens in this variant is due to a reduced amount of total Kell glycoprotein at the cell surface rather than to the inaccessibility of the antigens to Kell antibodies. Using a heterologous expression system, we demonstrate that the Kpa mutation causes retention of most of the Kell glycoprotein in a pre-Golgi compartment due to differential processing, thereby suggesting aberrant transport of the Kell protein to the cell surface. Furthermore, we demonstrated that single nucleotide substitutions into the coding region of the common KEL allele, as predicted by the molecular genotyping studies, was sufficient to encode three clinically significant low incidence antigens. We found that two low incidence antigens can be expressed on a single Kell protein, thus showing that the historical failure to detect such a variant is not due to structural constraints in the Kell protein. These studies demonstrate the power of studying the molecular mechanisms of blood group variants for elucidating the intracellular transport pathways of membrane proteins and the requirements for cell surface expression.
Alleles↗
Identification and characterization of novel human Ca(v)2.2 (alpha 1B) calcium channel variants lacking the synaptic protein interaction site.
The physical interaction between the presynaptic vesicle release complex and the large cytoplasmic region linking domains II and III of N-type (Ca(v)2.2) calcium channel alpha(1)B subunits is considered to be of fundamental importance for efficient neurotransmission. By PCR analysis of human brain cDNA libraries and IMR32 cell mRNA, we have isolated novel N-type channel variants, termed Ca(v)2.2-Delta1 and Delta2, which lack large parts of the domain II-III linker region, including the synaptic protein interaction site. They appear to be widely expressed across the human CNS as indicated by RNase protection assays. When expressed in tsA-201 cells, both novel variants formed barium-permeable channels with voltage dependences and kinetics for activation that were similar to those observed with the full-length channel. All three channel types exhibited the hallmarks of prepulse facilitation, which interestingly occurred independently of G-protein betagamma subunits. By contrast, the voltage dependence of steady-state inactivation seen with both Delta1 and Delta2 channels was shifted toward more depolarized potentials, and recovery from inactivation of Delta1 and Delta2 channels occurred more rapidly than that of the full-length channel. Moreover, the Delta1 channel was dramatically less sensitive to both omega-conotoxin MVIIA and GVIA than either the Delta2 variant or the full-length construct. Finally, the domain II-III linker region of neither variant was able to effectively bind syntaxin in vitro. These results suggest that the structure of the II-III linker region is an important determinant of N-type channel function and pharmacology. The lack of syntaxin binding hints at a unique physiological function of these channels.
Alternative Splicing↗
HARRIS AND WHITTAKER'S PSEUDOCHOLINESTERASE VARIANT WITH INCREASED RESISTANCE TO FLUORIDE. A STUDY OF FOUR FAMILIES AND THE IDENTIFICATION OF THE HOMOZGOTE.
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Ammonium Compounds↗
Identification of a splice-site mutation in the human growth hormone-variant gene.
The human growth-hormone-variant (hGH-V) gene normally expresses two alternatively spliced forms of mRNA--hGH-V and hGH-V2--in the placenta. hGH-V2 mRNA differs from hGH-V rDNA by the retention of intron 4 and represents approximately 15% of transcripts at term. In a survey of hGH-V gene expression in 20 placentas of gestational age 8-40 wk, we detected a single placenta that contained, in addition to the two normal hGH-V mRNA species, a set of two slightly larger hGH-V mRNAs. Sequence analysis of the elongated hGH-V mRNA demonstrated retention of the first 12 bases of intron 2, resulting from both a base substitution at the intron 2 splice-donor dinucleotide (GT----AT) and activation of a cryptic splice-donor site 12 bases downstream. Survey of a total of 60 additional chromosomes failed to reveal additional incidence of this mutation. The mutation, which we have designated hGH-Vintron 2, pos 1 (G----A), represents both an initial example of a nondeletional mutation within the hGH-V gene and corresponding structural alteration in the encoded hGH-V hormone.
Base Sequence↗
[Mycological picture index. 83. Variant formation of Trichophyton violaceum (Sabouraud) Bodin 1902 with reference to difficulties in identification of uncommon cultures of dermatophytes].
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Bacteriological Techniques↗
Characterization of N-terminal processing of group VIA phospholipase A2 and of potential cleavage sites of amyloid precursor protein constructs by automated identification of signature peptides in LC/MS/MS analyses of proteolytic digests.
Dysregulation of proteolytic processing of the amyloid precursor protein (APP) contributes to the pathogenesis of Alzheimer's Disease, and the Group VIA phospholipase A(2) (iPLA(2)beta) is the dominant PLA(2) enzyme in the central nervous system and is subject to regulatory proteolytic processing. We have identified novel N-terminal variants of iPLA(2)beta and previously unrecognized proteolysis sites in APP constructs with a C-terminal 6-myc tag by automated identification of signature peptides in LC/MS/MS analyses of proteolytic digests. We have developed a Signature-Discovery (SD) program to characterize protein isoforms by identifying signature peptides that arise from proteolytic processing in vivo. This program analyzes MS/MS data from LC analyses of proteolytic digests of protein mixtures that can include incompletely resolved components in biological samples. This reduces requirements for purification and thereby minimizes artifactual modifications during sample processing. A new algorithm to generate the theoretical signature peptide set and to calculate similarity scores between predicted and observed mass spectra has been tested and optimized with model proteins. The program has been applied to the identification of variants of proteins of biological interest, including APP cleavage products and iPLA(2)beta, and such applications demonstrate the utility of this approach.
Algorithms↗
Nuclear small subunit rRNA group I intron variation among Beauveria spp provide tools for strain identification and evidence of horizontal transfer.
An optional group I intron was characterized at a single insertion point in nuclear small subunit rRNA (nuSSU rRNA) genes of the imperfect entomopathogenic fungi, Beauveria bassiana and B. brongniartii. Insertion points were conserved among nuSSU rRNA genes from 35 Beauveria isolates. PCR-RFLP and DNA sequencing identified 12 group I intron variants and were applied to the identification of strains isolated from insect hosts. Alignment of 383-404-nt subgroup IB3 group I introns indicated that four insertion/deletion (indel) mutations were the main basis of fragment length variation. Phylogeny reconstruction using parsimony and neighbor-joining methods suggested six lineages may be present among nuSSU rRNA group I intron sequences from Beauveria and related ascomycete fungi. Terminal node placement of Beauveria introns conflicted with previously published phylogenies constructed from gene sequences, suggesting horizontal transfer of group I introns. PCR-RFLP among introns provided a means for the differentiation of Beauveria isolates.
Base Sequence↗
TRAIL-beta and TRAIL-gamma: two novel splice variants of the human TNF-related apoptosis-inducing ligand (TRAIL) without apoptotic potential.
Tumour necrosis factor (TNF) related apoptosis-inducing ligand (TRAIL/APO2L) is a recently identified member of the TNF family, which induces programmed cell death in a variety of neoplastic cell types, but not in most nonneoplastic cells. In this study, we report on the identification of two novel alternative splice variants of TRAIL in neoplastic and non-neoplastic human cells lacking either exon 3 (TRAIL-beta) or exons 2 and 3 (TRAIL-gamma). In both splice variants, loss of exon 3 resulted in a frame shift generating a stop codon with consecutive extensive truncation in the extracellular domain. Ectopic expression revealed a loss of proapoptotic potential for both alternative splice variants. In contrast to the predominantly cytoplasmatic localisation of GFP-tagged TRAIL-alpha and TRAIL-beta, TRAIL-gamma showed an additional association with the cell surface and nuclear membrane. In conclusion, alternative splicing might be involved in fine tuning of TRAIL-induced apoptosis and underlines the complexity of the TRAIL system.
Alternative Splicing↗
Isolation and characterization of a trisulfide variant of recombinant human growth hormone formed during expression in Escherichia coli.
A new variant of human growth hormone was recently found [Pavlu, B. & Gellerfors, P. (1993) Bioseparation 3, 257-265]. We report here the identification and the structural determination of this variant. The variant, which is formed during the expression of human growth hormone in Escherichia coli, was found to be more hydrophobic than rhGH as judged by its prolonged elution time by hydrophobic interaction chromatography. The rhGH hydrophobic variant (rhGH-HV) was isolated and subjected to trypsin digestion and RP-HPLC analysis, resulting in an altered retention time of one single tryptic peptide as compared to the corresponding fragment of rhGH. This tryptic peptide constitutes the C-terminus (aa 179-191) of hGH and contains one of the two disulfide bridges in hGH, viz. Cys182-Cys189. Amino acid sequences and composition analyses of the tryptic peptide from rhGH-HV (Tv18-19) and the corresponding tryptic peptide from rhGH (T18+19) were identical. Electrospray mass spectrometry (ES MS) of Tv18+19 isolated from rhGH-HV revealed a monoisotopic mass increase of 32.7, as compared to T18+19 from rhGH. A synthetic Tv18+19 peptide having a trisulfide bridge between Cys182 and Cys189 showed identical fragment in ES/MS compared to Tv18+19 isolated from rhGH-HV, i.e. m/z 617.7 and 682.9. These fragments are formed through a unique cleavage in the trisulfide (Cys182-SSS-Cys189) bridge not found in the corresponding T18+19 disulfide peptide. Furthermore, the synthetic Tv18+19 co-eluted in RP-HPLC with Tv18+19 isolated from rhGH-HV. Two-dimensional NMR spectroscopy of the synthetic T18+19 and Tv18+19 peptides were performed. Using these data all protons were assigned. The major chemical shift changes (delta delta > 0.05 ppm) observed were for the beta-protons of Cys182 and Cys189 in Tv18+19 as compared to T18+19. CD spectroscopy data were also in agreement with the above results. Based on these physico-chemical data rhGH-HV has been structurally defined as a trisulfide variant of rhGH. The receptor binding properties of rhGH-HV was studied by a biosensor device, BIAcore. The binding capacity of rhGH-HV was similar to rhGH with a binding stoichiometry to the rhGHBP of 1:1.6 and 1:1.5, respectively, indicating that the trisulfide modification did not affect its receptor binding properties.
Amino Acid Sequence↗
Identification and characterization of alternative splice products encoded by the human phosphotyrosyl phosphatase activator gene.
The phosphotyrosyl phosphatase activator (PTPA), a protein phosphatase 2A (PP2A) regulatory protein, specifically stimulates the phosphotyrosyl phosphatase activity of PP2A in vitro. Human PTPA is encoded by a single gene, the structure and chromosomal localization of which have been determined in our previous work. In this paper, we report the identification and characterization of six additional splice variants, termed PTPAbeta to PTPAeta, in addition to the originally identified PTPAalpha form. Interestingly, PTPAbeta and PTPAgamma contain a novel exon that had been overlooked in the formerly identified gene structure. As revealed by nested PCR, all these PTPA transcripts are expressed in various human cDNA libraries and cell lines. However, a quantitative approach, using a single PCR reaction followed by detection of the reaction products with a radioactively labeled probe, revealed only PTPAalpha, beta and delta, suggesting that the other transcripts are expressed very poorly. In vitro transcription-translation revealed that only PTPAalpha, beta, delta and epsilon are translated into functional proteins, whereas translation of PTPAgamma, zeta and eta is stopped prematurely due to a frameshift resulting from skipping exon 2, suggesting that the latter isoforms may result from splicing errors. By western analysis of HepG2 and Saos-2 cell extracts, only PTPAalpha and beta were detected. PTPAalpha and beta were expressed as GST fusion proteins in bacteria, and were found to contain the same specific phosphotyrosyl phosphatase stimulatory activity towards PP2A. The identification of this family of PTPA variants adds another level of complexity to the in vivo function(s) of PTPA, opening up the possibility that different isoforms may perform different functions.
Alternative Splicing↗
The relationship of cathepsin B and stefin A mRNA localization identifies a potentially aggressive variant of human prostate cancer within a Gleason histologic score.
Cathepsin B (CB) is involved in degradation of extracellular matrix proteins during tumor progression in human solid organ tumors (such as colorectal, bladder, and breast cancers), including human prostate cancer. Its activities are regulated by endogenous inhibitors (such as stefins or cystatins). Increased expression of cathepsin B message, protein, and membrane association have been linked to malignancy, but there are very few studies of their mRNA expression in prostate cancer using in situ hybridization techniques. Our objective was to determine the relationship of CB and stefin A (cystatin A) mRNA localization to the Gleason grading system for histologic scores in the hope of distinguishing aggressive and less aggressive variants of prostate cancer. We used a 25-base biotinylated oligonucleotide CB cDNA antisense probe to localize CB message and a 27-base biotinylated oligonucleotide stefin A cDNA antisense probe to localize stefin A message. Prostate samples from 41 prostatectomy patients were collected along with their pre-surgery serum PSA levels and clinical stage of the disease. Sections prepared from frozen prostate tissue samples were hybridized with the CB and stefin A and control pBR 322 probes using techniques reported by Sinha et al. [1] and their distribution quantitated by an image analysis system. Prostate sections treated with RNAse before hybridization or incubated with the pBR 322 control probe showed little or no reaction products, confirming that localization of CB and stefin A probes was specific. In prostate cancer, the reaction products were found in neoplastic and invasive cells and occasionally in stromal cells. The ratios of CB to stefin A were similar in normal prostate and benign prostatic hyperplasia (BPH) whereas they varied consistently within and between Gleason histologic scores for prostate cancer. These variations showed three localization patterns; namely, prostate cancers with higher levels of CB than stefin A, lower levels of CB than stefin A, and similar levels of CB and stefin A. All three patterns and ratios for CB and stefin A were found in prostate samples (22/41) represented by the Gleason histologic score 6 tumors. In these tumors, serum PSA levels ranged from 1 to 78 ng/ml and prostate cancers showed B, C, and D clinical stages. There was no correlation of CB/stefin A ratio and serum PSA values or clinical stage in a limited number of prostate cancer cases. Our data showed that there were prostate cancer cases within Gleason histologic scores which expressed high, similar, and low levels of CB when compared to stefin A. We postulate that prostate cancer cases showing higher levels of CB compared to stefin A probably represent an aggressive variant of this cancer within any one Gleason histologic score. If this is the case, aggressive variants of prostate cancer would occur within Gleason scores 3 to 10 even though higher scores are usually considered more aggressive forms of prostate cancers. Since our study is based upon a very limited number of frozen prostate samples, we emphasize that a larger series of archival prostate cancer samples along with their survival data should be analyzed to establish any relationship of CB/stefin A ratio and aggressive variants of this cancer. Therefore, our conclusion is tentative. Our study provides a partial explanation for differences in the clinical course of prostate cancer in patients. This is the first study to show that determination of CB and stefin A mRNA ratios may lead to identification of aggressive and less aggressive variants of prostate cancer within a Gleason histologic score.
Adenocarcinoma↗