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PubMed1968

Modified biochemical tests for characterization of L-phase variants of bacteria.

Biochemical capabilities of bacterial and L-phase organisms of 15 bacterial strains were examined by using a variety of modified routine diagnostic biochemical tests. The results demonstrated that 13 of the tests examined were suitable for use with L-phase variants, that L-phase variants and revertant bacterial phase organisms retained diagnostically significant capabilities of the respective bacterial or L-phase organisms from which they were derived, and that the 13 biochemical tests could be usefully employed to relate a given L-phase variant to a given bacterial phase organism, to distinguish L-phase variants of different species, and to aid in the identification of nonreverting L-phase variants.

Arginine↗
PubMed2005

Towards the identification of type II secretion signals in a nonacylated variant of pullulanase from Klebsiella oxytoca.

Pullulanase (PulA) from the gram-negative bacterium Klebsiella oxytoca is a 116-kDa surface-anchored lipoprotein of the isoamylase family that allows growth on branched maltodextrin polymers. PulA is specifically secreted via a type II secretion system. PelBsp-PulA, a nonacylated variant of PulA made by replacing the lipoprotein signal peptide (sp) with the signal peptide of pectate lyase PelB from Erwinia chrysanthemi, was efficiently secreted into the medium. Two 80-amino-acid regions of PulA, designated A and B, were previously shown to promote secretion of beta-lactamase (BlaM) and endoglucanase CelZ fused to the C terminus. We show that A and B fused to the PelB signal peptide can also promote secretion of BlaM and CelZ but not that of nuclease NucB or several other reporter proteins. However, the deletion of most of region A or all of region B, either individually or together, had only a minor effect on PelBsp-PulA secretion. Four independent linker insertions between amino acids 234 and 324 in PelBsp-PulA abolished secretion. This part of PulA, region C, could contain part of the PulA secretion signal or be important for its correct presentation. Deletion of region C abolished PelBsp-PulA secretion without dramatically affecting its stability. PelBsp-PulA-NucB chimeras were secreted only if the PulA-NucB fusion point was located downstream from region C. The data show that at least three regions of PulA contain information that influences its secretion, depending on their context, and that some reporter proteins might contribute to the secretion of chimeras of which they are a part.

Acylation↗
PubMed1991

Identification of a consistent pattern of mutations in neurovirulent variants derived from the sabin vaccine strain of poliovirus type 2.

Complete nucleotide sequencing of the RNAs of two unrelated neurovirulent isolates of Sabin-related poliovirus type 2 revealed that two nucleotides and one amino acid (amino acid 143 in the major capsid protein VP1) consistently departed from the sequences of the nonneurovirulent poliovirus type 2 712 and Sabin vaccine strains. This pattern of mutation appeared to be a feature common to all neurovirulent variants of poliovirus type 2.

Amino Acid Sequence↗
PubMed1998

Sample dilution to resolve mistaken identification of haemoglobin D as haemoglobin E using the variant automated system.

High performance liquid chromatography (HPLC) is increasingly being used to estimate variant haemoglobins. A case of haemoglobin S/D (HbS/D) is presented, which was misdiagnosed as haemoglobin S/E (HbS/E) by HPLC. The patient was a 22 year old woman with sickle cell anaemia. Subsequent haemodilution by blood transfusion clearly elucidated the haemoglobin D peak on HPLC. Sample dilution experiments, using the pretransfusion sample, were done resulting in correct elution of the peak in the D window. Troubleshooting in similar problematic haemoglobin variant peaks seen on HPLC can be done by sample dilution.

Adult↗
PubMed1985

Isolation and characterization of nuclear envelopes from three variant cell lines of the Shionogi mouse mammary carcinoma: identification of androgen-dependent peptides.

We have isolated and purified, with good yields, nuclear envelopes from an androgen-responsive and from two androgen-unresponsive cell lines of the Shionogi mouse mammary carcinoma after subjecting purified nuclei to DNase at high pH and characterized them morphologically, chemically, and enzymatically. Phase-contrast microscopy revealed the nuclei to be free of cytoplasmic tags and that the nuclear envelopes were isolated as membrane "ghosts." Electron micrographs clearly showed the double-membrane system with nuclear pore complexes which illustrates that the nuclear envelopes were ultrastructurally intact. The nuclear envelopes contained little DNA, low levels of arylesterase or acid phosphatase activity, and undetectable levels of succinate dehydrogenase and 5'-nucleotidase activity. Coomassie blue staining of the nuclear envelope fractions on sodium dodecyl sulfate-polyacrylamide gels for all three cell lines revealed that most of the polypeptides were similar. However, we have identified androgen-dependent peptides of molecular weights 29 000, 32 000, and 34 000 in nuclear envelopes of the androgen-responsive cell line peptide profiles by comparing the nuclear envelopes prepared from the androgen-responsive cell line grown in intact mice, in castrated mice, and in mice which had been injected with testosterone after castration. Further investigation of the androgen regulation of these nuclear envelope peptides may help us understand the molecular mechanisms involved during morphological changes of the nucleus which occur in response to different hormonal environments.

Acid Phosphatase↗
PubMed2005

Neural models for predicting viral vaccine targets.

We applied artificial neural networks (ANN) for the prediction of targets of immune responses that are useful for study of vaccine formulations against viral infections. Using a novel data representation, we developed a system termed MULTIPRED that can predict peptide binding to multiple related human leukocyte antigens (HLA). This implementation showed high accuracy in the prediction of the promiscuous peptides that bind to five HLA-A2 allelic variants. MULTIPRED is useful for the identification of peptides that bind multiple HLA-A2 variants as a group. By implementing ANN as a classification engine, we enabled both the prediction of peptides binding to multiple individual HLA-A2 molecules and the prediction of promiscuous binders using a single model. The ANN MULTIPRED predicts peptide binding to HLA-A*0205 with excellent accuracy (area under the receiver operating characteristic curve--AROC>0.90), and to HLA-A*0201, HLA-A*0204 and HLA-A*0206 with high accuracy (AROC>0.85). Antigenic regions with high density of binders ("antigenic hot-spots") represent best targets for vaccine design. MULTIPRED not only predicts individual 9-mer binders but also predicts antigenic hot spots. Two HLA-A2 hot-spots in Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) membrane protein were predicted by using MULTIPRED.

Algorithms↗
PubMed2006

Identification and functional characterization of a novel unspliced transcript variant of HIC-1 in human cancer cells exposed to adverse growth conditions.

The wild-type p53 gene has been widely implicated in the regulation of hypermethylated in cancer-1 (HIC-1) transcription, a master growth regulatory gene with multiple promoters and, consequently, multiple alternatively spliced transcripts. We investigated the role of p53 (wild type and mutant, both endogenous and exogenous) in modulating the various HIC-1 transcripts. We discovered a novel unspliced HIC-1 transcript, identified as "f" in leukocytes and in the human cell lines U87MG (wild-type p53), U373MG (mutant p53), MCF-7 (wild-type p53), HeLa (p53 degraded by HPV18-E6 oncoprotein), and Saos-2 (p53 null). This transcript is initiated from a new transcription start site and has an intervening stop codon that would result in a possibly truncated 22-amino-acid polypeptide. When U87MG (wild-type p53) and MCF-7 cells (wild-type p53) were exposed to adverse growth conditions of serum starvation or treated with the chemotherapeutic agent cisplatin, cells underwent apoptosis and cell cycle arrest accompanied by increase in p53 and HIC-1 transcript levels. Although the increase of the HIC-1-spliced transcripts followed the increase of p53, increase in f transcript coincided with declining p53 and HIC-1 transcript and protein levels. Moreover, the levels of HIC-1 f transcript were not induced by exogenously transfected wild-type p53 in p53-mutated U373MG and p53-null Saos-2 cells, unlike the spliced transcripts that code for full-length HIC-1 protein. These findings suggest a working model wherein the status of f transcript, which is not under direct transcriptional control of wild-type p53, may influence the level of HIC-1 protein in cancer cells.

Amino Acid Sequence↗
PubMed2000

Identification of wild-type and exon 5 deletion variants of estrogen receptor beta in normal human mammary gland.

We have examined messenger RNA (mRNA) expression of estrogen receptor (ER) alpha, wild-type ERbeta (mRNA and protein), and ERbeta exon 5 deletion variants (ERbeta delta5) in samples of normal human mammary gland obtained from 37 premenopausal subjects undergoing reduction mammoplasty. Comparing individual expression, ERbetaP mRNA predominated, expressed in 34 of 37 samples (91%), whereas ERalpha was found in 21 of 37 cases (57%). Receptor combinations were then analyzed and compared. Most samples either coexpressed ERalpha with ERbeta (54%) or expressed just ERbeta (38%). Immunohistochemical analysis revealed that ERbeta mRNA expression mirrored that of protein. Immunoreactivity was observed in the nucleus with additional evidence of cytoplasmic staining in those epithelial cells lining the breast ducts. Sporadic immunoreactivity was also detected in stromal cells. Expression of wild type and ERbeta delta5 was analyzed, and their association with ERalpha was compared. Most samples coexpressed wild-type ERbeta and the splice variant (62%; P = 0.05), with 30% exclusively expressing wild-type ERbeta. Although samples coexpressing wild type and variant ERbeta showed no statistical association with ERalpha, those samples expressing only wild-type ERP, showed a trend toward associations with ERalpha (P = 0.07). In conclusion, our data would support a role for ERbeta in the normal human mammary gland, where we propose it may be the dominant receptor.

Adolescent↗
PubMed2001

Identification of base substitutions in ten types of rare variants of phosphoglucomutase-1 (PGM1) encountered in Japanese.

In a previous starch-gel electrophoresis study of erythrocyte phosphoglucomutase-1 (PGM1) in 23,095 Japanese from Hiroshima and Nagasaki, we detected 14 types of rare variant alleles. To determine sequence differences in these rare alleles, cell lines were established from peripheral B-lymphocytes from 24 unrelated individuals in whom nine types of rare variants are presumed to exist on the basis of earlier electrophoresis studies. cDNAs reverse transcribed from mRNAs extracted from these cell lines were amplified by polymerase chain reaction and sequences determined. Amino acid substitution types were deduced from each cDNA sequence. Although two individuals were reported to have an identical electromorph (PGM1 4HR3), sequence analysis revealed that alleles encoding these electromorphs possessed different base substitutions, and one was renamed PGM1 4HR4. As the amino acid substitution of ten different variants could be deduced by cDNA sequence in this study, the effect of each amino acid substitution on enzyme activity could be precisely simulated. The secondary structure of each variant predicted by computer simulations revealed that very decreased activity observed on PGM1 4HR2 protein was caused by significant secondary structure change introduced by the amino acid substitution. On the basis of the crystal structure, the amino acid substitutions of the ten types of rare variants seem to be outside the active center of this enzyme.

Alleles↗
PubMed1997

Identification of G alpha s messenger ribonucleic acid splice variants in human granulosa cells.

Granulosa cells are essential for follicular development and corpus luteum formation and their functions are regulated by gonadotrophins through G protein-coupled receptors. The dominant second messenger pathway involves the stimulation of cyclic AMP formation by G alpha s-linked receptors. In this paper we have investigated the expression of G alpha s mRNA splice variants in relation to expression of G alpha s protein isoforms in granulosa cells obtained from patients undergoing in vitro fertilization. We have carried out ribonuclease protection assays using cRNA riboprobes which are capable of detecting all G alpha s mRNA isoforms as well as quantifying total amounts of G alpha s mRNA. Granulosa cells express the message for G alpha s-Large and G alpha s-Small and the presence of two distinct protein products was confirmed by immunoblotting using the antibody RM/1. Moreover, the data show that a significant fraction of G alpha s-Large and G alpha s-Small mRNAs contain an extra CAG codon. This should generate proteins with an extra serine residue, resulting in G alpha s variants with the consensus sequence of a protein kinase C phosphorylation site. These results highlight the possible interaction between different signalling pathways in the control of cAMP production and the need to investigate the relationship between G alpha s variants and different adenylyl cyclase isozymes in patients with normal and abnormal ovarian function.

Female↗
PubMedDate not supplied

Identification of human seminal plasma components that bind IgG2 variant.

Human seminal plasma (hsp) contains soluble proteins capable of binding immunoglobulin (Ig) G. Two novel components with estimated molecular sizes of 90 and 21 kD interact specifically with a variant of IgG2 found in 20% of human sera tested. The common IgG2 present in human sera and other subclasses of IgG did not bind with the hsp components. The present findings shows that the interacting IgG2 is a variant and not the common or prevalent species. The 90-kD component of hsp with IgG2 binding property is probably a nonglycosylated protein, whereas the 21-kD component is a glycosylated protein. The 90- and 21-kD components were detected in 20% of hsp specimens tested. Thus they are not present in the majority of hsp. Since the IgG2 binding components of hsp and the serum IgG2 variant are found in 20% of men and 20% of individuals, respectively, they can be used as genetic markers.

Antibodies, Monoclonal↗
PubMed1995

Application of capillary electrophoresis in the identification of phenotypes containing the beta-lactoglobulin C variant.

A method for the separation of the beta-LG variants A, B, and C was developed using free zone capillary electrophoresis. A separation buffer consisting of 50 mM 2-(N-morpholino)ethane sulfonic acid (pH 8.0) and polyoxyethylene (20)-sorbitan monolaurate was used for the separation. Milk samples from 424 Jersey cows were phenotyped using this method, and the gene frequencies for beta-LG A, B, and C were .41, .53, and .06, respectively. These frequencies were different from those found in studies of Jersey populations in other countries, where the beta-LG B allele had a significantly higher frequency, and the beta-LG A allele a significantly lower frequency, than those of this present study. The frequency of the beta-LG C allele was intermediate to those of other studies. The concentration of beta-LG in the milk was different among the beta-LG phenotypes and was, from greatest to least: AA, AB, AC, BB, and BC.

Animals↗
PubMed1986

Identification of a glycolipid precursor of the Trypanosoma brucei variant surface glycoprotein.

The variant surface glycoprotein (VSG) of Trypanosoma brucei has a glycolipid covalently attached to its C terminus. This glycolipid, which anchors the protein to the cell membrane, is attached to the VSG polypeptide within 1 min after translation (Bangs, J. D. Hereld, D., Krakow, J.L., Hart, G. W., and Englund, P. T. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 3207-3211). This rapid processing suggests that, prior to incorporation, the glycolipid may exist in the cell as a preformed precursor which is transferred to the VSG polypeptide en bloc. We have isolated a molecule which has properties consistent with it being a VSG glycolipid precursor. It is highly polar and can be labeled by [3H] myristate but not by [3H]palmitate. It reaches steady state during continuous labeling with [3H]myristate and shows rapid turnover in pulse-chase experiments, suggesting that it is a metabolic intermediate rather than an end product. When treated with HNO2 it liberates phosphatidylinositol, as does VSG (Ferguson, M. A. J., Low, M. G., and Cross, G. A. M. (1985) J. Biol. Chem. 260, 14547-14555). Also, like VSG, it releases a compound which co-migrates on thin layer chromatography with dimyristylglycerol when treated with the purified endogenous phospholipase C from trypanosomes. After treatment with this lipase, the putative precursor can be immunoprecipitated by antibodies directed against the C-terminal cross-reactive antigenic determinant of the VSG. These data provide strong evidence that this glycolipid is a VSG precursor.

Animals↗
PubMed1994

Identification of new mutations in two phosphoglycerate kinase (PGK) variants expressing different clinical syndromes: PGK Créteil and PGK Amiens.

Phosphoglycerate kinase (PGK) deficiency is generally associated with chronic hemolytic anemia, although it can be accompanied by either mental retardation or muscular disease. Genomic DNAs of two PGK-deficient patients previously described in France were sequenced directly after polymerase chain reaction amplification. The PGK Créteil variant arises from a G-->A nucleotide interchange at position 1022 in cDNA (exon 9), resulting in amino acid substitution 314 Asp-->Asn in the C-terminal domain, which contains the nucleotide binding site. It is associated with rhabdomyolysis crises but not with hemolysis or mental retardation. In the other case, which is associated with chronic hemolytic anemia and mental retardation (PGK Amiens), an A-->T nucleotide interchange was found at position 571 in cDNA (exon 5); this leads to amino acid substitution 163 Asp-->Val in the N-terminal domain, which contains the catalytic site for phosphoglycerate binding. These results corroborate the kinetic data observed. In the two cases, the mutations are distinct from others previously reported and no significant relationship could be observed between the location of the amino acid substitution and its clinical consequences.

Adult↗
PubMed2025

Creating an atlas of variant effects to resolve variants of uncertain significance and guide cardiovascular medicine.

Cardiovascular diseases are leading global causes of death and disability, often presenting as interrelated phenotypes of atherosclerotic vascular disease, heart failure and arrhythmias. Cardiovascular diseases arise from interactions between environmental factors and predisposing genotypes and include common Mendelian lipid disorders, cardiomyopathies and arrhythmia syndromes. The identification of a pathogenic variant through genetic testing can inform disease diagnosis, risk prediction, treatment and family screening. However, a major roadblock in genomic medicine is that for many variants, especially missense variants, we lack sufficient evidence to enable a definitive classification, and therefore these variants are deemed as 'variants of uncertain significance'. In this Review, we describe how multiplexed assays of variant effects can enable the functional assessment of nearly all coding variants in a target sequence, potentially offering a proactive approach to identifying the functional significance of gene variants that are observed later in a patient. We discuss validation, including the role of in silico variant effect predictors, and how multiplexed experimental methods are informing cardiovascular disease biology and ultimately resolving the problem of variants of uncertain significance at scale.

Humans↗
PubMed2001

Human Ca2+/calmodulin-dependent phosphodiesterase PDE1A: novel splice variants, their specific expression, genomic organization, and chromosomal localization.

We report here the identification of novel human PDE1A splice variants, their tissue distribution patterns, genomic structure, and chromosomal localization of the gene. We identified one N-terminus (N3) and one C-terminus (C3) by cDNA library screening and dbEST database search. These N- and C-termini, including the reported N-termini (N1 and N2) and C-termini (C1 and C2), combined to generate nine different PDE1A cDNAs. N1 and N2 are similar to the 5' ends of the bovine PDE1A proteins of 61 kDa and 59 kDa, respectively, and C1 and C2 are the 3' ends of the reported human PDE1A variants. The results of PCR and Southern blot analysis show that nine PDE1A splice variants exhibit distinctive tissue distribution patterns by the difference of the N-terminus. PDE1As with N2 were widely expressed in various tissues, mainly in the kidney, liver, and pancreas. On the other hand, PDE1As with N1 and N3 were particularly expressed at a high level in the brain and testis, respectively. These findings suggest that the distinct expression patterns among PDE1A variants depend on the several promoters situated upstream of exons encoding 5' ends of the variants. The PDE1A gene spans over 120 kb of genomic DNA, and consists of at least 17 exons and 16 introns. The PDE1A gene was located on human chromosome 2q32 by fluorescent in situ hybridization analysis.

Amino Acid Sequence↗
PubMed2006

RHD(T201R, F223V) cluster analysis in five different ethnic groups and serologic characterization of a new Ethiopian variant DARE, the DIII type 6, and the RHD(F223V).

BACKGROUND: The RHD phylogeny in humans shows four main clusters of which three are predominantly observed in (African) black persons. Each of the African clusters is characterized by specific amino acid substitutions relative to the Eurasian RHD allele. RH phylogeny defines the framework for identification of clinically relevant aberrant alleles. This study focuses on the weak D type 4 cluster (characterized by RHD(T201R, F223V) (602C>G 667T>G)) in five ethnic groups. STUDY DESIGN AND METHODS: A total of 1702 samples were screened for the presence of 602C>G and 667T>G by sequence-specific polymerase chain reaction (PCR-SSP). Eighty samples were assigned to the weak D type 4 cluster and were molecularly characterized by PCR-SSP and RHD sequencing. Antigens of aberrant alleles were characterized with monoclonal anti-D according to the 37-epitope model when possible. RESULTS: Five new aberrant alleles, DIII type 6, DIII type 7, DARE, RHD(T201R, F223V) (without 819G>A), and RHD(F223V), were identified and DIII type 6, DARE, and RHD(F223V) were serologically characterized with monoclonal anti-D. Both the DARE and RHD(F223V) showed epitope loss. It is postulated that the 1136C>T nucleotide substitution (characteristic for the DAU allele cluster) is present on the DVa(KOU) allele. CONCLUSION: Identification of the new variant alleles refines the phylogeny of RHD in humans. The proposed DVa(KOU) allele with 1136C>T (DVa(KOU)T379M) is probably caused by conversion of the DAU0 allele and the DVa(KOU) allele, forming a phylogenetic link between the DV allele and the DAU cluster. By describing the RHD(F223V) (602C>G) and RHD(T201R, F223V) (602C>G and 667T>G) alleles formal proof is given for the origin of the non-Eurasian cluster.

Amino Acid Substitution↗