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PubMed2003

Identification of grapevines infected with divergent variants of Grapevine virus A using variant-specific RT-PCR.

RT-PCRs, designed for the specific detection of molecular variants of Grapevine virus A (GVA), were applied to analysis of the virus from various grapevines and isolates recovered from these grapevines in Nicotiana benthamiana. Results of SSCP, cloning and sequencing revealed that the combination of these RT-PCR techniques permits reliable and rapid identification of grapevines mixed-infected with divergent variants of GVA. The results suggest that such grapevines are very common among GVA-infected grapevines in vineyards in South Africa.

Base Sequence↗

PubMed1991

Identification and characterization of a new member of the prolactin family, placental lactogen-I variant.

This report describes the identification and characterization of a new member of the placental prolactin (PRL) family, termed placental lactogen-I variant (PL-Iv). PL-Iv was isolated from medium conditioned by late gestation placental explants. Rat PL-Iv was found to be closely related to rat PL-I. Amino-terminal sequence analysis indicated that PL-Iv shared approximately 88% sequence identity with the amino terminus of PL-I. PL-Iv proteins cross-reacted with antiserum to recombinant mouse PL-I and PL-Iv mRNA hybridized with a PL-I cDNA. Multiple PL-I and PL-Iv species were present in placental cytosol. Despite the structural similarities between PL-I and PL-Iv, distinct differences were also evident. Antibodies generated to the amino-terminal 19 amino acids of PL-Iv specifically recognized PL-Iv, while failing to recognize PL-I. Secreted PL-Iv had an affinity for concanavalin A, whereas secreted PL-I lacked affinity for the lectin. PL-I was predominantly secreted as a 36-40-kDa species and PL-Iv was predominantly secreted as a 33-kDa species. Furthermore, PL-I and PL-Iv were synthesized at different times during gestation and by different cell types. PL-I was synthesized by trophoblast giant cells during the first half of gestation, while PL-Iv was predominantly synthesized by spongiotrophoblast cells during the later stages of gestation. PL-Iv was shown to stimulate the proliferation of rat Nb2 lymphoma cells, an in vitro measure of lactogenic activity. In summary, PL-Iv shares structural similarities with PL-I; however, it shows other structural differences in addition to unique cell- and temporal-specific patterns of expression in the rat chorioallantoic placenta.

Amino Acid Sequence↗

PubMed2004

[Identification and characterization of alternativly splicing variants for murine mater gene].

Mater encoding an oocyte-specific autoantigen,and is associated with premature autoimmune ovarian dysgenesis (AOD) in mouse. Based on RT-PCR, cDNA cloning, screening, sequencing and analysis, we have detected a total of four Mater splice variants, designated as variant B, E, F and G. All these splicing forms are in frame in terms of expected protein products. Among these, B was consistent with the previous report, whereas E, F, G belong to novel splice variants that have not been reported previously. Variant E lacks exon 6, variant F both lacks exon 10 and retains a part of intron 8, variant G lacks part of exon 14, and variant H lacks part of exon 13. The cDNA sequences at all the exon-intron boundaries confirms to the "GT-AG" splicing rule. Variant B, E, F exist in all the four strains. Variant G exists only in SWR/J. According to the cDNA sequences of these four splice variants, amimo acid sequences of the corresponding expected protein isoforms were deduced, and their potential functional effects were predicted in this thesis. Further identification and characterization of these expected protein isoforms would provide valuable information for their functional importance.

Alternative Splicing↗

PubMed1982

Biophysical identification and sorting of high metastatic variants from B16 melanoma tumor.

Chromatin structure, in terms of higher order nuclear-DNA condensation (scanning cytometry) and in terms of acridine orange primary binding sites (flow cytometry), is analyzed and shown to be significantly different between high (B16-F10) and low (B16-F1) metastatic variants of B16 melanoma. Furthermore, double staining of B16-F10 and B16-F1 with ethidium bromide (chromatin) and fluorescamine (membranes) provides the identification of a homogeneous subpopulation of cells with enhanced metastatic potential based on differential fluorescamine uptake. Fluorescamine uptake and poststaining viability is shown to be dependent upon the dye/cell ratio at which staining occurs. Utilizing a sterile cell sorting technique, a subpopulation of B16-F10 with increased fluorescamine uptake representing 30% of the total "intact cell" population was isolated by means of a fluorescence activated cell sorter and replated in vitro. This subpopulation when assayed in vivo produced significantly more pulmonary metastases than its parent cell line. Scanning cytometry of the Feulgen stained sorted subpopulation reveals that the cells possess a unique nuclear morphometry characterized by a 2C-3C DNA content and a large nuclear area (disperse chromatin). Finally, when we assay simultaneously for nuclear-DNA organization and cell membrane organization a progressive uncoupling between nuclear and cell morphometry is apparent if B16-F10 (versus B16-F1).

Animals↗

PubMed1996

Characterization of dentatorubral-pallidoluysian atrophy proteins using two-dimensional electrophoretic analysis.

The genetic defect dentatorubral-pallidoluysian atrophy (DRPLA) is known to be an expansion of a CAG trinucleotide repeat. The mutant gene has been demonstrated to be translated into protein, and this protein contains an expanded polyglutamine region. In the present study, we have demonstrated using two-dimensional gel electrophoresis and immunoblotting that the DRPLA gene products consist of a number of isoelectric variants in brain tissue and lymphoblastoid cells. The identification of isoelectric variants indicates that there may be multiple stages of post-translational modification. DRPLA proteins from brain tissue possess fewer basic isoelectric variants than those from lymphoblastoid cells, suggesting that there is different post-translational modification steps in brain tissue and lymphoblastoid cell.

Adolescent↗

PubMed2004

Human CYP4F12 genetic polymorphism: identification and functional characterization of seven variant allozymes.

The human cytochrome CYP4F12 has been shown to be metabolically active toward inflammatory mediators and exogenous compounds such as antihistaminic drugs. We recently identified a genetic polymorphism within the promoter region, associated with a decreased level of enzyme expression. In the present study, we report the further identification of single nucleotide polymorphisms in the coding sequence of the CYP4F12 gene. A polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis of DNA samples from 53 unrelated French Caucasians, allowed the identification of ten mutations, comprising seven missense mutations, 31C>T (Leu11Phe), 38C>T (Pro13Leu), 47C>T (Met16Thr), 4759G>A (Asp76Asn), 4801G>A (Val90Leu), 8896C>T (Arg188Cys) and 23545G>A (Gly522Ser). Their functional impact toward ebastine hydroxylation was evaluated using heterologous expression in Saccharomyces cerevisiae cells of site-directed mutated cDNA variants. Five out seven variants did not exhibit any significant difference in CYP4F12 catalytic activity, whereas two variants, Val90Ile and Arg188Cys, displayed significant changes in their Michaelis-Menten (Km, Vm) parameters. These data on CYP4F12 genetic polymorphism provide tools for further studies of association with pathological processes involving an inflammatory component and with variations in anti-histaminic drug response.

Aryl Hydrocarbon Hydroxylases↗

PubMed1994

Electrophoretic screening for genetic variation in apolipoprotein C-III: identification of a novel apoC-III variant, apoC-III(Asp45-->Asn), in a Turkish patient.

Screening of 6,840 plasma samples by isoelectric focusing (IEF) led to the identification of a novel apolipoprotein C-III variant. The underlying molecular defect was established by sequencing of exons 3 and 4 of the apoC-III gene subsequent to their amplification by the polymerase chain reaction (PCR). A G-->A transition in the first nucleotide of codon 45 results in a replacement of aspartic acid by asparagine. ApoC-III(Asp45-->Asn) was detected in a Turkish patient who previously had undergone coronary bypass surgery. Family studies identified two of the three children of the index patient as heterozygous variant carriers. The family was too small to demonstrate a significant effect of the variant on lipid metabolism. However, as judged by two-dimensional immunoelectrophoresis as well as IEF and subsequent scanning densitometry, the concentrations of the variant allele products were increased twofold in very low density lipoproteins (VLDL) and slightly decreased both in low density lipoproteins (LDL) and in high density lipoproteins (HDL) relative to the concentrations of the normal allele products. The disproportional distribution of the variant apoC-III isoproteins may indicate differences in the metabolism of variant and normal apoC-III. We conclude that genetically determined structural variants of apoC-III with changes in complete net charges are very rare and, hence, do not significantly contribute to the formation of dyslipidemia in the German population. Although heterozygosity for apoC-III(Asp45-->Asn) is not associated with severe dyslipidemia, the disproportional distribution of the allele products among plasma lipoproteins indirectly indicates some impact on lipoprotein metabolism.

Alleles↗

PubMed2002

Genetics of insulin resistance.

Insulin resistance, defined as the decreased ability of insulin to perform its biological functions, is likely to represent the primary physiologic defect underlying the insulin resistance syndrome (IRS), which includes insulin resistance/hyperinsulinemia, glucose intolerance and/or type 2 diabetes mellitus, visceral obesity, hypertension, and dyslipidemia. This constellation of traits is a leading cause of cardiovascular mortality and morbidity. Insulin sensitivity varies widely among individuals. Although environmental provocations including physical inactivity and caloric excess play an important role in the development of obesity and thus insulin resistance, epidemiologic and family studies show that there are also moderate genetic influences on the development of insulin resistance. Extreme forms of insulin resistance may be caused rarely by mutations in the genes for the insulin receptor and peroxisome proliferator-activated receptor gamma. However, the genetic basis for common more moderate forms of insulin resistance is likely to be polygenic and heterogeneous. Evidence further suggests that gene variants may have phenotypic influences on more than one IRS trait (so-called pleiotrophy), which may explain, in part, the clustering of these traits. This article reviews the evidence that insulin resistance has a genetic basis. Progress to date toward identifying specific gene variants are reviewed. Ultimately, the identification of specific gene variants that influence insulin resistance and other IRS traits will have profound influences on our understanding of the molecular and pathophysiologic basis of these disorders, from which new and more effective preventive and therapeutic interventions will be possible.

Chromosomes, Human, Pair 19↗

PubMed2006

Insight into the complex genetic network of tetraploid Atlantic salmon (Salmo salar L.): description of multiple novel Pax-7 splice variants.

Paired box transcription factor 7 (Pax-7) cDNA was isolated from the skeletal muscle and brain of alevin and adult stages of Atlantic salmon, identifying 10 variants categorised as novel or established insertions (ins) or deletions (del). Two putative Pax-7 paralogs were identified (denoted Pax-7alpha and Pax-7beta) on the basis of the length and sequences of intron 3 (218 and 248 bp) and versions of ins1 and ins2. Pax-7beta contained a threonine variant of ins1 (GQY[T]GPEYVYCGT), and a shortened variant of ins2 (GEAS). Pattern identification revealed the threonine variant of ins1 includes a potential phosphorylation site (casein kinase II). Thus, the tetraploid Atlantic salmon genome appears to contain at least two putative copies and multiple splice variants of Pax-7. In situ hybridisation localised Pax-7 to mononuclear cells in the fast muscle of adult Atlantic salmon, while quantitative real-time PCR showed Pax-7alpha to be more highly expressed in brain than in skeletal muscle.

Amino Acid Sequence↗

PubMed2004

Genetic typing of shiga toxin 2 variants of Escherichia coli by PCR-restriction fragment length polymorphism analysis.

Shiga toxins Stx1 and Stx2 play a prominent role in the pathogenesis of Shiga toxin-producing Escherichia coli (STEC) infections. Several variants of the stx(2) gene, encoding Stx2, have been described. In this study, we developed a PCR-restriction fragment length polymorphism system for typing stx(2) genes of STEC strains. The typing system discriminates eight described variants and allows the identification of new stx(2) variants and STEC isolates carrying multiple stx(2) genes. A phylogenetic tree, based on the nucleotide sequences of the toxin-encoding genes, demonstrates that stx(2) sequences with the same PvuII HaeIII HincII AccI type generally cluster together.

Animals↗

PubMed2001

Papillary carcinoma of the thyroid and its variants: a cytohistological correlation.

Fine-needle aspiration cytology (FNAC) is considered highly specific for the diagnosis of papillary carcinoma of the thyroid (PCT). In recent years, several variants of PCT have been described. An attempt was made to gauge the accuracy of classification of variants of PCT on aspirates. Cytology smears from 124 of 150 cases of histologically proven PCT with a prior FNAC were reviewed over a 16-yr period. A diagnosis of papillary carcinoma on FNAC was made in 93 cases. Further subclassification of these cases was done on cytology and tissue sections independently. The variants of PCT classified on FNAC were classical PCT (PCT-CL), 76 cases; Hurthle-cell variant (PCT-HCV), 3 cases; follicular variant (PCT-FV), 6 cases; tall-cell variant (PCT-TCV), 2 cases; high-grade variant (PCT-HG), 2 cases; and 2 cases each which were debatably PCT-CL/PCT-FV and PCT-CL/PCT-HG. Cytology typing was accurate in 65 of the 72 classical variants, while only 7 of the 22 follicular variants were correctly identified on cytology. Two of the 3 high-grade papillary carcinomas (PCT-HG) were identified on FNAC, and the solitary case of tall-cell variant could readily be classified on cytology. In conclusion, identification of the various variants of PCT is possible, though difficulty is encountered in correctly categorizing the follicular variant, which is often mistaken for a follicular neoplasm. Also, identification of the solid variant and the papillary carcinoma with nodular fasciitis-like stroma was a problem on cytology. Another interesting observation in our series is that an admixture of various cell types was seen in the smears and corroborated on histology to be present in focal areas.

Biopsy, Needle↗

PubMed2000

Identification of specific BRCA1 and BRCA2 variants by DHPLC.

Denaturing high performance liquid chromatography (DHPLC) is generating increasing interest in clinical genetics as a reliable tool for the analysis of genetic alterations. In the work presented here our intentions were to optimize primer design and DHPLC analysis conditions for a qualitative detection of BRCA1 and BRCA2 variations. The BRCA1 and BRAC2 genes display a high proportion of polymorphisms. Sequencing efforts geared towards the distinction of tumor-related mutations and benign variants still remain time-consuming and expensive. DHPLC elution profiles, however, permit the correlation of a characteristic chromatographic profile with a specific sequence alteration. In this study we evaluate the sensitivity of DHPLC for the identification of unique polymorphisms, which are frequent in the Caucasian population, in lieu of sequence analysis. The complete BRCA1 gene and parts of BRCA2 were examined. In the case of BRCA1, 431 out of 432 heterozygotes were identified correctly. In addition, 18 new profiles were identified which had not been detected previously in our studies and which represented new mutations or rare polymorphisms. For BRCA2, 135 out of 137 simple sequence variants were classified correctly. In addition, six new profiles were identified which represented new mutations or rare polymorphisms.

BRCA2 Protein↗

PubMed1985

[Identification with roseofungin of an antibiotic produced by Streptomyces roseoflavus strain A-23/791 and its undifferentiated variant].

Isolation, purification and chemical identification of an antibiotic produced by strain IMV A-23/791 of Streptomyces roseoflavus and its adifferentiated variant are described. The antibiotic inhibits the growth of many phytopathogenic fungi, dermatophytes and yeast-like fungi and is capable of stimulating the growth and development of plants. The antibiotic is effective in control of cucumber root rot under hydroponic cultivation conditions. It was shown that the adifferentiated proactinomycetous variant 4-76 was more productive than the initial strain A-23/791. The antibiotic isolated from the variant mycelium is more active. It was shown that the antibiotic can be isolated from the biomass of the adifferentiated variant with a more simple method in the authors' modification providing higher antibiotic yields and the use of lower amounts of the solvents. The UV, IR, mass and luminescence spectra and mobility in paper and thin-layer chromatography have shown the carbonyl-conjugated pentaene A-23/791 to be identical to roseofungin described earlier. Based on the data obtained strain A-23/791 should be classified with Streptomyces roseoflavus var, roseofungini Nikitina, 1968, a variant of Streptomyces roseoflavus Arai, 1951.

Antifungal Agents↗

PubMedDate not supplied

[Genome polymorphism in dissociative variants of Bacillus subtilis (mesentericus) 76. Identification of dissociants using genome fingerprinting].

DNA fingerprinting procedure with M13 repeat probe as we have shown earlier makes it possible to apply a new approach in theoretical and applied fields of microbiology and bacteriology. In this work, using the method described we have revealed genomic polymorphism of dissociative variants of Bac. subtilis (mesentericus) 76. The data obtained may be referred as strong evidence that bacterial dissociation do has genetic nature.

Bacillus subtilis↗

PubMed1979

Comparison of chloramphenicol acetyltransferase variants in staphylococci. Purification, inhibitor studies and N-terminal sequences.

Four electrophoretic variants of chloramphenicol acetyltransferase (types A, B, C and D) found in chloramphenicol-resistant staphylococci were purified by affinity chromatography. Michaelis constants and the kinetics of inactivation with a variety of reagents for the four variants are virtually identical. Their similar amino acid compositions and near identical N-terminal sequences suggest a high degree of overall sequence homology. The thiol-specific reagents 5,5'-dithiobis-(2-nitrobenzoic acid), 2-nitro-5-thiocyanobenzoic acid and 2,2'-dithiopyridine are without significant effect on enzyme activity, whereas 1-fluoro-2,4-dinitrobenzene, N-ethylmaleimide, p-chloromercuribenzoic acid, iodoacetamide, and, particularly, bromoacetyl-CoA and diethyl pyrocarbonate are potent inhibitors. Iodoacetate is not an inhibitor. The results of chemical modification studies on the four enzyme variants and the identification of 3-carboxymethylhistidine in acid hydrolysates of one variant (type C) after inactivation with iodoacetamide suggest that a unique histidine residue may be involved in the mechanism of catalysis.

Acetyltransferases↗

PubMed2004

Identification and molecular characterization of Mnk1b, a splice variant of human MAP kinase-interacting kinase Mnk1.

In this paper, we report the identification and molecular characterization of a splice variant of human Mnk1 which has been named as Mnk1b. Human Mnk1b mRNA is homologous to human Mnk1 mRNA but lacking a region corresponding to exon 19, which causes a change in the reading frame generating a stop codon. The resulting protein lacks the last 89 amino acids at the C-terminal region that are replaced by 12 amino acids with an entirely new sequence. The C-terminal end in Mnk1 corresponds to the extracellular signal-regulated kinase (ERK1/2) binding site. Although Mnk1b lacks this domain and, consequently, is not phosphorylated by ERK1/2, it is able, however, to phosphorylate eIF4E in vitro and in vivo in a mitogen-activated protein kinases-independent manner. This result suggests that Mnk1b may play a key role in regulating protein translation in the absence of stimuli. Interestingly, a significant population of cells shows Mnk1b within the nucleus whereas Mnk1 is always detected in the cytoplasm. This fact may be explained because Mnk1b maintains the nuclear localization signal (NLS) but lacks the nuclear export sequence (NES).

Alternative Splicing↗

PubMed1998

Melanocortin 1 receptor variants in an Irish population.

The identification of an association between variants in the human melanocortin 1 receptor (MC1R) gene and red hair and fair skin, as well as the relation between variants of this gene and coat color in animals, suggests that the MC1R is an integral control point in the normal pigmentation phenotype. In order to further define the contribution of MC1R variants to pigmentation in a normal population, we have looked for alterations in this gene in series of individuals from a general Irish population, in whom there is a preponderance of individuals with fair skin type. Seventy-five per cent contained a variant in the MC1R gene, with 30% containing two variants. The Arg151Cys, Arg160Trp, and Asp294His variants were significantly associated with red hair (p = 0.0015, p < 0.001, and p < 0.005, respectively). Importantly, no individuals harboring two of these three variants did not have red hair, although some red-haired individuals only showed one alteration. The same three variants were also over-represented in individuals with light skin type as assessed using a modified Fitzpatrick scale. Despite these associations many subjects with dark hair/darker skin type harbored MC1R variants, but there was no evidence of any particular association of variants with the darker phenotype. The Asp294His variant was similarly associated with red hair in a Dutch population, but was infrequent in red-headed subjects from Sweden. The Asp294His variant was also significantly associated with nonmelanoma skin cancer in a U.K. population. The results show that the Arg151Cys, Arg160Trp, and Asp294His variants are of key significance in determining the pigmentary phenotype and response to ultraviolet radiation, and suggest that in many cases the red-haired component and in some cases fair skin type are inherited as a Mendelian recessive.

Adult↗

PubMed2001

Maturation of rat brain is accompanied by differential expression of the long and short splice variants of G(s)alpha protein: identification of cytosolic forms of G(s)alpha.

Distribution of the alpha subunit of the stimulatory G protein (G(s)alpha) was analyzed in membrane and cytosolic (supernatant 200 000 g) fractions from rat cortex, thalamus and hippocampus during the course of post-natal development. In parallel, changes in beta-adrenoceptor density and adenylyl cyclase activity were determined. Long (G(s)alphaL) and short (G(s)alphaS) variants of G(s)alpha were assessed by immunoblotting using specific polyclonal antisera reacting with both G(s)alpha isoforms. Post-natal development was associated with an increase in the total amount of brain G(s)alpha. G(s)alphaL was the dominant isoform of G(s)alpha in the membrane fractions of all studied brain regions and its amount increased markedly between post-natal day (PD) 1 and 90. The level of membrane-bound G(s)alphaS also elevated during post-natal development, but more pronounced changes were found in cytosolic G(s)alphaS. Although only a small amount of G(s)alphaS (much smaller than G(s)alphaL) was detected among soluble proteins shortly after birth, G(s)alphaS prevailed over G(s)alphaL at PD90. The G(s)alphaL/G(s)alphaS ratio decreased, respectively, from 3.2 to 1.2 and from 5.0 to 1.5 in the membrane fractions of cortex and hippocampus, but remained almost constant in thalamus between PD1 and 90. More dramatic changes were found in the cytosolic fractions of all studied brain regions: the G(s)alphaL/G(s)alphaS ratio decreased sharply in cortex (from 14.1 to 0.9), hippocampus (from 3.7 to 0.8), and also in thalamus (from 9.5 to 0.5). These results demonstrate that the membrane-cytosol balance of G(s)alpha proteins alters dramatically during the course of brain development. Both G(s)alphaL and G(s)alphaS were expressed in a region- and age-specific manner, which suggests different roles in the maturation of the brain tissue. A cyc(-) reconstitutive assay of cytosolic G(s)alpha indicated that only approximately 20% of this protein was functional, compared with membrane-bound G(s)alpha, and its ability to reconstitute adenylyl cyclase activity increased during the course of maturation. The number of beta-adrenoceptors increased sharply during early post-natal development but only slightly in adulthood, and both GTP- and isoproterenol-stimulated adenylate cyclase activity reached peak values around PD12.

Adenylyl Cyclases↗