scholarly journals A Human FSHB Promoter SNP Associated With Low FSH Levels in Men Impairs LHX3 Binding and Basal FSHB Transcription

Endocrinology ◽  
2013 ◽  
Vol 154 (9) ◽  
pp. 3016-3021 ◽  
Author(s):  
Courtney A. Benson ◽  
Troy L. Kurz ◽  
Varykina G. Thackray
Keyword(s):  
Untranslated Region ◽  
The Other ◽  
Hormone Response ◽  
Potential Mechanism ◽  
Wild Type ◽  
Nucleotide Polymorphism ◽  
Transcription Start ◽  
Single Nucleotide ◽  
Hormone Response Element ◽  
Inactivating Mutations

FSH production is important for human gametogenesis. In addition to inactivating mutations in the FSHB gene, which result in infertility in both sexes, a G/T single-nucleotide polymorphism (SNP) at −211 relative to the transcription start site of the 5′ untranslated region of FSHB has been reported to be associated with reduced serum FSH levels in men. In this study, we sought to identify the potential mechanism by which the −211 SNP reduces FSH levels. Although the SNP resides in a putative hormone response element, we showed that, unlike the murine gene, human FSHB was not induced by androgens or progestins in gonadotropes. On the other hand, we found that the LHX3 homeodomain transcription factor bound to an 11-bp element in the human FSHB promoter that includes the −211 nucleotide. Furthermore, we also demonstrated that LHX3 bound with greater affinity to the wild-type human FSHB promoter compared with the −211 G/T mutation and that LHX3 binding was more effectively competed with excess wild-type oligonucleotide than with the SNP. Finally, we showed that FSHB transcription was decreased in gonadotrope cells with the −211 G/T mutation compared with the wild-type FSHB promoter. Altogether, our results suggest that decreased serum FSH levels in men with the SNP likely result from reduced LHX3 binding and induction of FSHB transcription.

Analysis of Additional Virulence Genes and Virulence Gene Regions in Listeria monocytogenes Confirms the Epidemiologic Relevance of Multi-Virulence-Locus Sequence Typing

2008 ◽  
Vol 71 (12) ◽  
pp. 2559-2566 ◽  
Author(s):  
SARA LOMONACO ◽  
YI CHEN ◽  
STEPHEN J. KNABEL
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Listeria Monocytogenes ◽  
Virulence Genes ◽  
Virulence Gene ◽  
The Other ◽  
Evolutionary Divergence ◽  
Gene Sequences ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Molecular Subtyping

Previous molecular subtyping studies have defined four epidemic clones (ECs) of Listeria monocytogenes (ECI, ECII, ECIII, and ECIV). Partial sequences of eight virulence genes were previously shown to be identical within individual ECs of L. monocytogenes. The present study was conducted to determine if the sequences of other virulence genes and virulence gene regions are also conserved within these ECs. Six additional virulence genes—bsh, hly, inlJ, lplA1, pgdA, and srtA—and three additional virulence gene regions of actA, inlA, and inlB were selected based on their role in L. monocytogenes virulence, and intragenic regions of each gene were sequenced. Sequencing was performed on a diverse set of 44 to 48 L. monocytogenes strains. Results demonstrated that the sequenced regions of the nine virulence genes were identical within each of the ECs, and 257 new single nucleotide polymorphism (SNPs) were identified. ECIII (lineage II) was easily distinguishable from the other ECs, as 238 SNPs were observed in ECIII due to its significant evolutionary divergence from lineage I. With regard to the other ECs, there were 5 SNPs that represented an informative set, since these SNPs were able to differentiate specific ECs from all other unrelated strains used in this study. This study confirms our previous finding that virulence gene sequences are highly conserved within individual ECs and contain stable SNPs that can be used to very accurately differentiate ECs of L. monocytogenes from each other and from other diverse strains.

scholarly journals Determination of the Factor V Leiden Single-Nucleotide Polymorphism in a Commercial Clinical Laboratory by Use of NanoChip Microelectronic Array Technology

2002 ◽  
Vol 48 (9) ◽  
pp. 1406-1411 ◽  
Author(s):  
Jess G Evans ◽  
Cindy Lee-Tataseo
Keyword(s):  
Clinical Laboratory ◽  
Factor V Leiden ◽  
Factor V ◽  
Third Wave ◽  
Wild Type ◽  
Wave Analysis ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
The Third ◽  
Array Technology

Abstract Background: Methods for analysis of the single-nucleotide polymorphism (SNP) known as factor V Leiden (FVL) are described. The technique provides rapid, highly accurate detection of the point mutation that encodes for replacement of arginine-506 with glutamine. After formal assay qualification, 758 clinical samples that had previously been analyzed by the InvaderTM Monoplex Assay were tested as research samples in a commercial clinical laboratory. Methods: Primers specific for factor V (FV) were prepared, and PCR was performed. Samples were analyzed using the NanoChip® Molecular Biology Workstation with fluorescently labeled reporters for wild-type and SNP sequences. Results: Of the 635 samples classified by the Third WaveTM assay as FV wild type, 10 were identified as heterozygous FVL by the NanoChip technique. Similarly, of the 114 putative heterozygous samples, 4 were wild type, and of the 9 reported homozygous samples, 6 were homozygous, 2 were heterozygous, and 1 was FV wild type by the NanoChip assay. All 17 results that were discordant with the Third Wave analysis were confirmed by DNA sequencing to be correctly classified by the NanoChip technology. The Nanochip system was 100% accurate in characterizing wild-type, heterozygous, and homozygous samples compared with accuracies of 99.2%, 90.2%, and 100% for the comparable Third Wave analysis. Conclusions: The NanoChip microelectronic chip array technology is an accurate and convenient method for FVL screening of research samples in a clinical laboratory environment.

scholarly journals Bottom-up single-molecule strategy for understanding subunit function of tetrameric β-galactosidase

2018 ◽  
Vol 115 (33) ◽  
pp. 8346-8351 ◽  
Author(s):  
Xiang Li ◽  
Yu Jiang ◽  
Shaorong Chong ◽  
David R. Walt
Keyword(s):  
Single Molecule ◽  
Wild Type ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Single Molecule Level ◽  
Subunit Function ◽  
Enzyme Molecules ◽  
Individual Enzyme ◽  
Kinetics Of

In this paper, we report an example of the engineered expression of tetrameric β-galactosidase (β-gal) containing varying numbers of active monomers. Specifically, by combining wild-type and single-nucleotide polymorphism plasmids at varying ratios, tetrameric β-gal was expressed in vitro with one to four active monomers. The kinetics of individual enzyme molecules revealed four distinct populations, corresponding to the number of active monomers in the enzyme. Using single-molecule-level enzyme kinetics, we were able to measure an accurate in vitro mistranslation frequency (5.8 × 10−4 per base). In addition, we studied the kinetics of the mistranslated β-gal at the single-molecule level.

scholarly journals Serotonin 5-HT2C Receptor Cys23Ser Single Nucleotide Polymorphism Associates with Receptor Function and Localization In Vitro

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Michelle A. Land ◽  
Holly L. Chapman ◽  
Brionna D. Davis-Reyes ◽  
Daniel E. Felsing ◽  
John A. Allen ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Subcellular Localization ◽  
Cell Lines ◽  
Intracellular Signaling ◽  
Calcium Release ◽  
Wild Type ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Recycling Pathway

Abstract A non-synonymous single nucleotide polymorphism of the human serotonin 5-HT2C receptor (5-HT2CR) gene that converts a cysteine to a serine at amino acid codon 23 (Cys23Ser) appears to impact 5-HT2CR pharmacology at a cellular and systems level. We hypothesized that the Cys23Ser alters 5-HT2CR intracellular signaling via changes in subcellular localization in vitro. Using cell lines stably expressing the wild-type Cys23 or the Ser23 variant, we show that 5-HT evokes intracellular calcium release with decreased potency and peak response in the Ser23 versus the Cys23 cell lines. Biochemical analyses demonstrated lower Ser23 5-HT2CR plasma membrane localization versus the Cys23 5-HT2CR. Subcellular localization studies demonstrated O-linked glycosylation of the Ser23 variant, but not the wild-type Cys23, may be a post-translational mechanism which alters its localization within the Golgi apparatus. Further, both the Cys23 and Ser23 5-HT2CR are present in the recycling pathway with the Ser23 variant having decreased colocalization with the early endosome versus the Cys23 allele. Agonism of the 5-HT2CR causes the Ser23 variant to exit the recycling pathway with no effect on the Cys23 allele. Taken together, the Ser23 variant exhibits a distinct pharmacological and subcellular localization profile versus the wild-type Cys23 allele, which could impact aspects of receptor pharmacology in individuals expressing the Cys23Ser SNP.

Single nucleotide polymorphism in prohibitin 3′untranslated region and breast-cancer susceptibility

The Lancet ◽  
2001 ◽  
Vol 357 (9268) ◽  
pp. 1588-1589 ◽  
Author(s):  
Eldon R Jupe ◽  
Allen A Badgett ◽  
Barbara R Neas ◽  
Melissa A Craft ◽  
Debra S Mitchell ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Single Nucleotide Polymorphism ◽  
Untranslated Region ◽  
Cancer Susceptibility ◽  
Breast Cancer Susceptibility ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide

scholarly journals Novel Genotyping and Quantitative Analysis of Neuraminidase Inhibitor Resistance-Associated Mutations in Influenza A Viruses by Single-Nucleotide Polymorphism Analysis

2011 ◽  
Vol 55 (10) ◽  
pp. 4718-4727 ◽  
Author(s):  
Susu Duan ◽  
David A. Boltz ◽  
Jiang Li ◽  
Christine M. Oshansky ◽  
Henju Marjuki ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Influenza Viruses ◽  
Polymorphism Analysis ◽  
Snp Analysis ◽  
Pandemic H1n1 ◽  
Wild Type ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Inhibitor Resistance ◽  
Public Health Epidemiology

ABSTRACTNeuraminidase (NA) inhibitors are among the first line of defense against influenza virus infection. With the increased worldwide use of the drugs, antiviral susceptibility surveillance is increasingly important for effective clinical management and for public health epidemiology. Effective monitoring requires effective resistance detection methods. We have developed and validated a novel genotyping method for rapid detection of established NA inhibitor resistance markers in influenza viruses by single nucleotide polymorphism (SNP) analysis. The multi- or monoplex SNP analysis based on single nucleotide extension assays was developed to detect NA mutations H275Y and I223R/V in pandemic H1N1 viruses, H275Y in seasonal H1N1 viruses, E119V and R292K in seasonal H3N2 viruses, and H275Y and N295S in H5N1 viruses. The SNP analysis demonstrated high sensitivity for low-content NA amplicons (0.1 to 1 ng/μl) and showed 100% accordant results against a panel of defined clinical isolates. The monoplex assays for the H275Y NA mutation allowed precise and accurate quantification of the proportions of wild-type and mutant genotypes in virus mixtures (5% to 10% discrimination), with results comparable to those of pyrosequencing. The SNP analysis revealed the lower growth fitness of an H275Y mutant compared to the wild-type pandemic H1N1 virus by quantitatively genotyping progeny viruses grown in normal human bronchial epithelial cells. This novel method offers high-throughput screening capacity, relatively low costs, and the wide availability of the necessary equipment, and thus it could provide a much-needed approach for genotypic screening of NA inhibitor resistance in influenza viruses.

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