scholarly journals Regulation of the Sox3 Gene in an X0/X0 Mammal without Sry, the Amami Spiny Rat, Tokudaia osimensis

2019 ◽  
Vol 159 (3) ◽  
pp. 143-150
Author(s):  
Kohei Washio ◽  
Shusei Mizushima ◽  
Takamichi Jogahara ◽  
Asato Kuroiwa
Keyword(s):  
Sex Determination ◽  
Reporter Gene ◽  
Promoter Region ◽  
Sex Chromosome ◽  
Fold Increase ◽  
Specific Pattern ◽  
Sox9 Expression ◽  
Mouse Sequence ◽  
Gene Assay ◽  
Sox3 Gene

Two species of spiny rats, Tokudaia osimensis and Tokudaia tokunoshimensis, show an X0/X0 sex chromosome constitution due to the lack of a Y chromosome. The Sry gene has been completely lost from the genome of these species. We hypothesized that Sox3, which is thought to be originally a homologue of Sry, could function in sex determination in these animals in the absence of Sry. Sox3 was localized in a region of the X chromosome in T. osimensis homologous to mouse. A similar testis- and ovary-specific pattern of expression was observed in mouse and T. osimensis. Although the sequence of the Sox3 gene and its promoter are highly conserved, a 13-bp deletion was specifically found in the promoter region of the 2 spiny rat species. Reporter gene assays were performed to examine the effect of the 13-bp deletion in the promoter region on Sox3 regulation. Although an approximately 60% decrease in activity was observed using the Tokudaia promoters with the 13-bp deletion, the activity was recovered using a mutated promoter in which the deletion was filled with mouse sequence. To evaluate whether SOX3 could regulate Sox9 expression, a reporter gene assay was carried out using testis-specific enhancer of Sox9 core (TESCO). Co-transfection with a combination of mouse SF1 and mouse SOX3 or T. osimensis SOX3 resulted in a greater than 2-fold increase in activity of mouse and T. osimensis TESCO. These results support the idea that the function of SOX3 as a transcription factor, as has been reported in mice and humans, is conserved in T. osimensis. Therefore, we conclude that the Sox3 gene has no function in sex determination in Sry-lacking Tokudaia species.

scholarly journals Functional identification of regulatory elements within the promoter region of platelet-derived growth factor 2.

1989 ◽  
Vol 9 (2) ◽  
pp. 396-405 ◽  
Author(s):  
M Pech ◽  
C D Rao ◽  
K C Robbins ◽  
S A Aaronson
Keyword(s):  
Growth Factor ◽  
Reporter Gene ◽  
Promoter Region ◽  
Promoter Activity ◽  
K562 Cells ◽  
Fold Increase ◽  
Regulatory Elements ◽  
Platelet Derived Growth Factor ◽  
Regulatory Sequence ◽  
Mrna Levels

Human platelet-derived growth factor (PDGF) is composed of two polypeptide chains, PDGF-1 and PDGF-2, the human homolog of the v-sis oncogene. Deregulation of PDGF-2 expression can confer a growth advantage to cells possessing the cognate receptor and, thus, may contribute to the malignant phenotype. We investigated the regulation of PDGF-2 mRNA expression during megakaryocytic differentiation of K562 cells. Induction by 12-O-tetradecanoylphorbol-13-acetate (TPA) led to a greater than 200-fold increase in PDGF-2 transcript levels in these cells. Induction was dependent on protein synthesis and was not enhanced by cycloheximide exposure. In our initial investigation of the PDGF-2 promoter, a minimal promoter region, which included sequences extending only 42 base pairs upstream of the TATA signal, was found to be as efficient as 4 kilobase pairs upstream of the TATA signal in driving expression of a reporter gene in uninduced K562 cells. We also functionally identified different regulatory sequence elements of the PDGF-2 promoter in TPA-induced K562 cells. One region acted as a transcriptional silencer, while another region was necessary for maximal activity of the promoter in megakaryoblasts. This region was shown to bind nuclear factors and was the target for trans-activation in normal and tumor cells. In one tumor cell line, which expressed high PDGF-2 mRNA levels, the presence of the positive regulatory region resulted in a 30-fold increase in promoter activity. However, the ability of the minimal PDGF-2 promoter to drive reporter gene expression in uninduced K562 cells and normal fibroblasts, which contained no detectable PDGF-2 transcripts, implies the existence of other negative control mechanisms beyond the regulation of promoter activity.

Functional identification of regulatory elements within the promoter region of platelet-derived growth factor 2

1989 ◽  
Vol 9 (2) ◽  
pp. 396-405
Author(s):  
M Pech ◽  
C D Rao ◽  
K C Robbins ◽  
S A Aaronson
Keyword(s):  
Growth Factor ◽  
Reporter Gene ◽  
Promoter Region ◽  
Promoter Activity ◽  
K562 Cells ◽  
Fold Increase ◽  
Regulatory Elements ◽  
Platelet Derived Growth Factor ◽  
Regulatory Sequence ◽  
Mrna Levels

Human platelet-derived growth factor (PDGF) is composed of two polypeptide chains, PDGF-1 and PDGF-2, the human homolog of the v-sis oncogene. Deregulation of PDGF-2 expression can confer a growth advantage to cells possessing the cognate receptor and, thus, may contribute to the malignant phenotype. We investigated the regulation of PDGF-2 mRNA expression during megakaryocytic differentiation of K562 cells. Induction by 12-O-tetradecanoylphorbol-13-acetate (TPA) led to a greater than 200-fold increase in PDGF-2 transcript levels in these cells. Induction was dependent on protein synthesis and was not enhanced by cycloheximide exposure. In our initial investigation of the PDGF-2 promoter, a minimal promoter region, which included sequences extending only 42 base pairs upstream of the TATA signal, was found to be as efficient as 4 kilobase pairs upstream of the TATA signal in driving expression of a reporter gene in uninduced K562 cells. We also functionally identified different regulatory sequence elements of the PDGF-2 promoter in TPA-induced K562 cells. One region acted as a transcriptional silencer, while another region was necessary for maximal activity of the promoter in megakaryoblasts. This region was shown to bind nuclear factors and was the target for trans-activation in normal and tumor cells. In one tumor cell line, which expressed high PDGF-2 mRNA levels, the presence of the positive regulatory region resulted in a 30-fold increase in promoter activity. However, the ability of the minimal PDGF-2 promoter to drive reporter gene expression in uninduced K562 cells and normal fibroblasts, which contained no detectable PDGF-2 transcripts, implies the existence of other negative control mechanisms beyond the regulation of promoter activity.

scholarly journals In vitro bioanalytical assessment of toxicity of wetland samples from Spanish Mediterranean coastline

2021 ◽  
Vol 33 (1) ◽  
Author(s):  
Alberto Celma ◽  
Geeta Mandava ◽  
Agneta Oskarsson ◽  
Juan Vicente Sancho ◽  
Lubertus Bijlsma ◽  
...  
Keyword(s):  
Water Quality ◽  
Reporter Gene ◽  
Biological Activities ◽  
Reporter Gene Assay ◽  
Water Bodies ◽  
Good Tool ◽  
Adverse Health Effects ◽  
Aryl Hydrocarbon ◽  
Gene Assay

Abstract Background Fresh water bodies represent less than 1% of overall amount of water on earth and ensuring their quality and sustainability is pivotal. Although several campaigns have been performed to monitor the occurrence of micropollutants by means of chemical analysis, this might not cover the whole set of chemicals present in the sample nor the potential toxic effects of mixtures of natural and anthropogenic chemicals. In this sense, by selecting relevant toxicity endpoints when performing in vitro bioanalysis, effect-based methodologies can be of help to perform a comprehensive assessment of water quality and reveal biological activities relevant to adverse health effects. However, no prior bioanalytical study was performed in wetland water samples from the Spanish Mediterranean coastline. Methods Eleven samples from relevant water bodies from the Spanish Mediterranean coastline were collected to monitor water quality on 8 toxicity endpoints. Aryl hydrocarbon receptor (AhR), androgenicity (AR+ and AR−), estrogenicity (ER+ and ER−), oxidative stress response (Nrf2) and vitamin D receptor (VDR+ and VDR−) reporter gene assays were evaluated. Results AhR was the reporter gene assay showing a more frequent response over the set of samples (activated by 9 out of 11 samples), with TCDD-eq in the range 7.7–22.2 pM. For AR, ER and VDR assays sporadic activations were observed. Moreover, no activity was observed on the Nrf2 reporter gene assay. Wastewater and street runaway streams from Valencia could be responsible for enhanced activities in one of the water inputs in the Natural Park ‘L’Albufera’. Conclusions Water quality of relevant wetlands from the Spanish Mediterranean coastline has been evaluated. The utilization of a panel of 5 different bioassays to cover for different toxicity endpoints has demonstrated to be a good tool to assess water quality.

scholarly journals The Diversity and Evolution of Sex Chromosomes in Frogs

Genes ◽  
2021 ◽  
Vol 12 (4) ◽  
pp. 483
Author(s):  
Wen-Juan Ma ◽  
Paris Veltsos
Keyword(s):  
Sex Determination ◽  
Sex Chromosomes ◽  
Chromosome Evolution ◽  
Sex Chromosome ◽  
Comparative Genomic ◽  
Ancestral State ◽  
Heteromorphic Sex Chromosomes ◽  
Genomic Studies ◽  
Evolutionary Trajectories ◽  
Heterogametic Sex

Frogs are ideal organisms for studying sex chromosome evolution because of their diversity in sex chromosome differentiation and sex-determination systems. We review 222 anuran frogs, spanning ~220 Myr of divergence, with characterized sex chromosomes, and discuss their evolution, phylogenetic distribution and transitions between homomorphic and heteromorphic states, as well as between sex-determination systems. Most (~75%) anurans have homomorphic sex chromosomes, with XY systems being three times more common than ZW systems. Most remaining anurans (~25%) have heteromorphic sex chromosomes, with XY and ZW systems almost equally represented. There are Y-autosome fusions in 11 species, and no W-/Z-/X-autosome fusions are known. The phylogeny represents at least 19 transitions between sex-determination systems and at least 16 cases of independent evolution of heteromorphic sex chromosomes from homomorphy, the likely ancestral state. Five lineages mostly have heteromorphic sex chromosomes, which might have evolved due to demographic and sexual selection attributes of those lineages. Males do not recombine over most of their genome, regardless of which is the heterogametic sex. Nevertheless, telomere-restricted recombination between ZW chromosomes has evolved at least once. More comparative genomic studies are needed to understand the evolutionary trajectories of sex chromosomes among frog lineages, especially in the ZW systems.

scholarly journals Creating a human-induced pluripotent stem cell-based NKX2.5 reporter gene assay for developmental toxicity testing

2021 ◽  
Author(s):  
Karin Lauschke ◽  
Andreas Frederik Treschow ◽  
Mikkel Aabech Rasmussen ◽  
Nichlas Davidsen ◽  
Bjørn Holst ◽  
...  
Keyword(s):  
Stem Cell ◽  
Reporter Gene ◽  
Luminescence Intensity ◽  
Developmental Toxicity ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Luciferase Reporter ◽  
Reporter Cell ◽  
Gene Assay ◽  
Induced Pluripotent

AbstractTo test large numbers of chemicals for developmental toxicity, rapid in vitro tests with standardized readouts for automated data acquisition are needed. However, the most widely used assay, the embryonic stem cell test, relies on the counting of beating embryoid bodies by visual inspection, which is laborious and time consuming. We previously developed the PluriBeat assay based on differentiation of human induced pluripotent stem cells (hiPSC) that we demonstrated to be predictive for known teratogens at relevant concentrations using the readout of beating cardiomyocytes. Here, we report the development of a novel assay, which we term the PluriLum assay, where we have introduced a luciferase reporter gene into the locus ofNKX2.5of our hiPSC line. This enabled us to measure luminescence intensities instead of counting beating cardiomyocytes, which is less labor intensive. We established twoNKX2.5reporter cell lines and validated their pluripotency and genetic stability. Moreover, we confirmed that the genetically engineeredNKX2.5reporter cell line differentiated into cardiomyocytes with the same efficiency as the original wild-type line. We then exposed the cells to valproic acid (25–300 μM) and thalidomide (0.1–36 µM) and compared the PluriBeat readout of the cardiomyocytes with the luminescence intensity of the PluriLum assay. The results showed that thalidomide decreased luminescence intensity significantly with a higher potency and efficacy compared to the beating readout. With this, we have developed a novel hiPSC-based assay with a standardized readout that may have the potential for higher throughput screening for developmental toxicity.

scholarly journals Evolutionary transitions between mechanisms of sex determination in vertebrates

2011 ◽  
Vol 7 (3) ◽  
pp. 443-448 ◽  
Author(s):  
Alexander E. Quinn ◽  
Stephen D. Sarre ◽  
Tariq Ezaz ◽  
Jennifer A. Marshall Graves ◽  
Arthur Georges
Keyword(s):  
Sex Determination ◽  
Chromosome Pair ◽  
Sex Chromosome ◽  
Molecular Pathways ◽  
Genetic Contribution ◽  
Temperature Dependent ◽  
Trait Evolution ◽  
Evolutionary Transitions ◽  
Dichotomous Outcome ◽  
Threshold Trait

Sex in many organisms is a dichotomous phenotype—individuals are either male or female. The molecular pathways underlying sex determination are governed by the genetic contribution of parents to the zygote, the environment in which the zygote develops or interaction of the two, depending on the species. Systems in which multiple interacting influences or a continuously varying influence (such as temperature) determines a dichotomous outcome have at least one threshold. We show that when sex is viewed as a threshold trait, evolution in that threshold can permit novel transitions between genotypic and temperature-dependent sex determination (TSD) and remarkably, between male (XX/XY) and female (ZZ/ZW) heterogamety. Transitions are possible without substantive genotypic innovation of novel sex-determining mutations or transpositions, so that the master sex gene and sex chromosome pair can be retained in ZW–XY transitions. We also show that evolution in the threshold can explain all observed patterns in vertebrate TSD, when coupled with evolution in embryonic survivorship limits.

Evaluation of estrogenic activities of pesticides using an in vitro reporter gene assay

2005 ◽  
Vol 15 (4) ◽  
pp. 271-280 ◽  
Author(s):  
Mihoko Kojima ◽  
Kenji Fukunaga * ◽  
Mari Sasaki ◽  
Masafumi Nakamura ◽  
Motohiro Tsuji ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Reporter Gene Assay ◽  
Gene Assay

Weak male-determining genes and female heterogamety in Chironomus tentans

1984 ◽  
Vol 26 (6) ◽  
pp. 748-751
Author(s):  
Ray Feraday
Keyword(s):  
Sex Determination ◽  
Sex Chromosome ◽  
Chironomus Tentans ◽  
Female Development ◽  
Female Heterogamety ◽  
Dominant Female

Female heterogamety in the midge Chironomus tentans has been previously reported and attributed to a dominant female determiner. Published results are not consistent with the interpretation, and the female heterogamety, if any, can be better explained by a model involving a weakened male determiner. Suggestions are made for crosses between populations with different sex-determining mechanisms that would discriminate between models for the evolution of female heterogamety, and serve to determine whether indeed female development is the norm in the absence of any parental sex chromosomes.Key words: Chironomus, heterogamety, sex determination, sex chromosome.

Development of a novel reporter gene assay to evaluate antibody-dependent cellular phagocytosis for anti-CD20 therapeutic antibodies

2021 ◽  
Vol 100 ◽  
pp. 108112
Author(s):  
Chunyu Liu ◽  
Chuanfei Yu ◽  
Yalan Yang ◽  
Jing Huang ◽  
Xiaojuan Yu ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Reporter Gene Assay ◽  
Therapeutic Antibodies ◽  
Gene Assay ◽  
Anti Cd20

A supernumerary “B-sex” chromosome drives male sex determination in the Pachón cavefish, Astyanax mexicanus

2021 ◽  
Author(s):  
Boudjema Imarazene ◽  
Kang Du ◽  
Séverine Beille ◽  
Elodie Jouanno ◽  
Romain Feron ◽  
...  
Keyword(s):  
Sex Determination ◽  
Sex Chromosome ◽  
Astyanax Mexicanus ◽  
Male Sex
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