scholarly journals Cloning, Molecular Characterization and Expression Patterns of DMRTC2 Implicated in Germ Cell Development of Male Tibetan Sheep

2020 ◽  
Vol 21 (7) ◽  
pp. 2448 ◽  
Author(s):  
Taotao Li ◽  
Hongyu Zhang ◽  
Xia Wang ◽  
De′en Yin ◽  
Nana Chen ◽  
...  
Keyword(s):  
Amino Acids ◽  
Western Blot ◽  
Molecular Characterization ◽  
Expression Patterns ◽  
Testicular Development ◽  
Reading Frame ◽  
Tibetan Sheep ◽  
Pcr Method ◽  
Conserved Gene ◽  
First Time

The double sex and mab-3-related transcription factors like family C2 (DMRTC2) gene is indispensable for mammalian testicular function and spermatogenesis. Despite its importance, what expression and roles of DMRTC2 possesses and how it regulates the testicular development and spermatogenesis in sheep, especially in Tibetan sheep, remains largely unknown. In this study, DMRTC2 cDNA from testes of Tibetan sheep was firstly cloned by the RT-PCR method, and its molecular characterization was identified. Subsequently, the expression and localization patterns of DMRTC2 were evaluated by quantitative real-time PCR (qPCR), Western blot, and immunofluorescence. The cloning and sequence analysis showed that the Tibetan sheep DMRTC2 cDNA fragment contained 1113 bp open reading frame (ORF) capable of encoding 370 amino acids, and displayed high identities with some other mammals, which shared an identical DM domain sequence of 47 amino acids ranged from residues 38 to 84. qPCR and Western blot results showed that DMRTC2 was expressed in testes throughout the development stages while not in epididymides (caput, corpus, and cauda), with higher mRNA and protein abundance in Tibetan sheep testes of one- and three-year-old (post-puberty) compared with that of three-month-old (pre-puberty). Immunofluorescence results revealed that immune staining for DMRTC2 protein was observed in spermatids and spermatogonia from post-puberty Tibetan sheep testes, and gonocytes from pre-puberty Tibetan sheep testes. Together, these results demonstrated, for the first time, in sheep, that DMRTC2, as a highly conserved gene in mammals, is essential for sheep spermatogenesis by regulating the proliferation or differentiation of gonocytes and development of spermatids in ram testes at different stages of maturity.

scholarly journals Characterization of GLOD4 in Leydig Cells of Tibetan Sheep During Different Stages of Maturity

Genes ◽  
2019 ◽  
Vol 10 (10) ◽  
pp. 796 ◽  
Author(s):  
Wang ◽  
Li ◽  
Liu ◽  
Zhang ◽  
Zhao ◽  
...  
Keyword(s):  
Leydig Cells ◽  
Structural Characteristics ◽  
Sequence Similarity ◽  
Expression Patterns ◽  
Proteome Analysis ◽  
Testicular Development ◽  
Protein Levels ◽  
Tibetan Sheep ◽  
Pcr Method ◽  
One Year

We have previously reported that glyoxalase domain-containing protein 4 (GLOD4) is expressed in sheep testes by proteome analysis, but its roles during testicular development remain unclear. The aim of this study was to understand the expression characteristics and biological functions of the GLOD4 gene in developmental Tibetan sheep testes. The cDNA sequence of the Tibetan sheep GLOD4 gene was cloned by the RT-PCR method, and the structural characteristics of the GLOD4 protein were analyzed using relevant bioinformatics software, including ProtParam, TMHMM, Signal P 4.1, SOPMA, and phyre2. The expression patterns and immunolocalization of GLOD4 were examined in developmental testes derived from three-month-old (3M), one-year-old (1Y), and three-year-old (3Y) Tibetan sheep using quantitative real-time PCR (qRT-PCR), Western blot, immunohistochemistry, and immunofluorescence staining. The sequence analysis showed that the coding sequence (CDS) region of the GLOD4 gene was 729 bp in length and encoded 242 amino acids. Bioinformatics analysis found that the nucleotide and amino acid sequence of Tibetan sheep GLOD4 exhibited the highest sequence similarity with goat and chiru, and the least with zig-zag eel, of the species compared. GLOD4 expressions at both the mRNA and protein levels were significantly higher in the testes of the 1Y and 3Y groups than those in the 3M group (p < 0.01). Immunohistochemistry and immunofluorescence results indicated that the GLOD4 protein was mainly localized in the cytoplasm of Leydig cells from Tibetan sheep testes throughout the development stages. These results taken together suggest that the GLOD4 gene may be implicated in the development of the Leydig cells of Tibetan sheep during different stages of maturity.

scholarly journals Torque Teno Virus (SANBAN Isolate) ORF2 Protein Suppresses NF-κB Pathways via Interaction with IκB Kinases

2007 ◽  
Vol 81 (21) ◽  
pp. 11917-11924 ◽  
Author(s):  
Hong Zheng ◽  
Linbai Ye ◽  
Xiaonan Fang ◽  
Baozong Li ◽  
Yuhua Wang ◽  
...  
Keyword(s):  
Western Blot ◽  
Nuclear Factor Κb ◽  
Torque Teno Virus ◽  
Dependent Manner ◽  
Nuclear Factor ◽  
Innate And Adaptive Immunity ◽  
Reading Frame ◽  
Orf2 Protein ◽  
First Time ◽  
Dose Dependent

ABSTRACT Since the first discovery of Torque teno virus (TTV) in 1997, many researchers focused on its epidemiology and transcriptional regulation, but the function of TTV-encoded proteins remained unknown. The function of the TTV open reading frame (ORF) in the nuclear factor κB (NF-κB) pathway has not yet been established. In this study, we found for the first time that the TTV ORF2 protein could suppress NF-κB activity in a dose-dependent manner in the canonical NF-κB pathway. By Western blot analysis, we proved that the TTV ORF2 protein did not alter the level of NF-κB expression but prevented the p50 and p65 subunits from entering the nucleus due to the inhibition of IκBα protein degradation. Further immunoprecipitation assays showed that the TTV ORF2 protein could physically interact with IKKβ as well as IKKα, but not IKKγ. Luciferase assays and Western blot experiments showed that the TTV ORF2 protein could also suppress NF-κB activity in the noncanonical NF-κB pathway and block the activation and translocation of p52. Finally, we found that the TTV ORF2 protein inhibited the transcription of NF-κB-mediated downstream genes (interleukin 6 [IL-6], IL-8, and COX-2) through down-regulation of NF-κB. Together, these data indicate that the TTV ORF2 protein suppresses the canonical and noncanonical NF-κB pathways, suggesting that the TTV ORF2 protein may be involved in regulating the innate and adaptive immunity of organisms, contributing to TTV pathogenesis, and even be related to some diseases.

scholarly journals Quantitative and dynamic expression profile of premature and active forms of the regional ADAM proteins during chicken brain development

2011 ◽  
Vol 16 (3) ◽  
Author(s):  
Annett Markus ◽  
Xin Yan ◽  
Arndt Rolfs ◽  
Jiankai Luo
Keyword(s):  
Western Blot ◽  
Expression Patterns ◽  
Transmembrane Proteins ◽  
Tissue Formation ◽  
Chicken Brain ◽  
Dynamic Expression ◽  
Quantitative Western Blot ◽  
First Time ◽  
The Brain ◽  
Different Parts

AbstractThe ADAM (A Disintegrin and Metalloprotease) family of transmembrane proteins plays important roles in embryogenesis and tissue formation based on their multiple functional domains. In the present study, for the first time, the expression patterns of the premature and the active forms of six members of the ADAM proteins — ADAM9, ADAM10, ADAM12, ADAM17, ADAM22 and ADAM23 — in distinct parts of the developing chicken brain were investigated by quantitative Western blot analysis from embryonic incubation day (E) 10 to E20. The results show that the premature and the active forms of various ADAM proteins are spatiotemporally regulated in different parts of the brain during development, suggesting that the ADAMs play a very important role during embryonic development.

scholarly journals Phospholipase Cζ, the trigger of egg activation in mammals, is present in a non-mammalian species

Reproduction ◽  
2005 ◽  
Vol 130 (2) ◽  
pp. 157-163 ◽  
Author(s):  
K Coward ◽  
C P Ponting ◽  
H-Y Chang ◽  
O Hibbitt ◽  
P Savolainen ◽  
...  
Keyword(s):  
Amino Acids ◽  
Phospholipase C ◽  
Functional Properties ◽  
Mammalian Species ◽  
Open Reading Frame ◽  
Domestic Chicken ◽  
Egg Activation ◽  
Specific Gene ◽  
Reading Frame ◽  
First Time

The activation of the egg to begin development into an embryo is triggered by a sperm-induced increase in intracellular egg Ca2+. There has been much controversy about how the sperm induces this fundamental developmental event, but recent studies suggest that, in mammals, egg activation is triggered by a testis-specific phospholipase C: PLCζ. Since the discovery of PLCζ, it has been unclear whether its role in triggering egg activation is common to all vertebrates, or is confined to mammals. Here, we demonstrate for the first time that PLCζ is present in a non-mammalian vertebrate. Using genomic and cDNA databases, we have identified the cDNA encoding a PLCζ orthologue in the domestic chicken that, like the mammalian isoforms, is a testis-specific gene. The chicken PLCζ cDNA is 2152 bp in size and encodes an open reading frame of 639 amino acids. When injected into mouse oocytes, chicken PLCζ cRNA triggers Ca2+ oscillations, indicating that it has functional properties similar to those of mammalian PLCζ. Our findings suggest that PLCζ may have a universal role in triggering egg activation in vertebrates.

scholarly journals Differential Expression of Three α-Galactosidase Genes and a Single β-Galactosidase Gene from Aspergillus niger

1999 ◽  
Vol 65 (6) ◽  
pp. 2453-2460 ◽  
Author(s):  
Ronald P. de Vries ◽  
Hetty C. van den Broeck ◽  
Ester Dekkers ◽  
Paloma Manzanares ◽  
Leo H. de Graaff ◽  
...  
Keyword(s):  
Amino Acids ◽  
Aspergillus Niger ◽  
Signal Sequence ◽  
Expression Patterns ◽  
Carbon Sources ◽  
Gene Encoding ◽  
Calculated Molecular Mass ◽  
Reading Frame ◽  
Polymeric Compounds ◽  
Elevated Expression

ABSTRACT A gene encoding a third α-galactosidase (AglB) fromAspergillus niger has been cloned and sequenced. The gene consists of an open reading frame of 1,750 bp containing six introns. The gene encodes a protein of 443 amino acids which contains a eukaryotic signal sequence of 16 amino acids and seven putative N-glycosylation sites. The mature protein has a calculated molecular mass of 48,835 Da and a predicted pI of 4.6. An alignment of the AglB amino acid sequence with those of other α-galactosidases revealed that it belongs to a subfamily of α-galactosidases that also includesA. niger AglA. A. niger AglC belongs to a different subfamily that consists mainly of prokaryotic α-galactosidases. The expression of aglA,aglB, aglC, and lacA, the latter of which encodes an A. niger β-galactosidase, has been studied by using a number of monomeric, oligomeric, and polymeric compounds as growth substrates. Expression of aglA is only detected on galactose and galactose-containing oligomers and polymers. The aglB gene is expressed on all of the carbon sources tested, including glucose. Elevated expression was observed on xylan, which could be assigned to regulation via XlnR, the xylanolytic transcriptional activator. Expression of aglC was only observed on glucose, fructose, and combinations of glucose with xylose and galactose. High expression of lacA was detected on arabinose, xylose, xylan, and pectin. Similar to aglB, the expression on xylose and xylan can be assigned to regulation via XlnR. All four genes have distinct expression patterns which seem to mirror the natural substrates of the encoded proteins.

scholarly journals Unraveling Stage-Dependent Expression Patterns of Circular RNAs and Their Related ceRNA Modulation in Ovine Postnatal Testis Development

2021 ◽  
Vol 9 ◽  
Author(s):  
Taotao Li ◽  
Ruirui Luo ◽  
Xia Wang ◽  
Huihui Wang ◽  
Xingxu Zhao ◽  
...  
Keyword(s):  
Germ Cells ◽  
Sertoli Cells ◽  
Leydig Cells ◽  
Expression Patterns ◽  
Interaction Network ◽  
Functional Enrichment ◽  
Circular Rnas ◽  
Testicular Development ◽  
Tibetan Sheep ◽  
Igf1 Expression

Circular RNAs (circRNAs) have been shown to function in the reproductive systems including testis. However, their expression, as well as function in testicular development of sheep remain undefined. Herein, we performed RNA sequencing to reveal circRNA temporal expression patterns in testes of Tibetan sheep from different stages of maturation (3M, 3-month-old; 1Y, 1-year-old; 3Y, 3-year-old). A total of 3,982, 414, and 4,060 differentially expressed (DE) circRNAs were uncovered from 3M vs 1Y, 1Y vs 3Y, and 3M vs 3Y, respectively. Functional enrichment assessment indicated that the source genes of DE circRNAs were primarily engaged in spermatogenesis and testicular immune privilege including blood–testis barrier (BTB). We subsequently constructed the core circRNA–miRNA–mRNA interaction network for genes related to testicular function, such as spermatogenesis, germ cell development, BTB, and cell cycle/meiosis. Furthermore, we validated the target associations between either circ_024949, circ_026259 or IGF1, and oar-miR-29b in this network, and revealed their similar expression signatures in developmental testes that they were extensively expressed in germ cells, Leydig cells, and Sertoli cells, thus suggesting their broad functions in the functional maintenance of Leydig cells and Sertoli cells, as well as the development and maturation of male germ cells. Meanwhile, circ_026259 was shown to promote IGF1 expression through inhibition of oar-miR-29b in sheep Sertoli cells. This work gives the first global view for the expression and regulation of circRNAs in sheep testis, which will be helpful for providing new insights into the molecular mechanism of ovine testis function.

Molecular Analysis of EMS-Induced frizzled Mutations in Drosophila melanogaster

Genetics ◽  
1996 ◽  
Vol 142 (1) ◽  
pp. 205-215 ◽  
Author(s):  
Katherine H Jones ◽  
Jingchun Liu ◽  
P N Adler
Keyword(s):  
Amino Acids ◽  
Drosophila Melanogaster ◽  
Membrane Protein ◽  
Western Blot ◽  
Normal Tissue ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Tissue Polarity ◽  
Sds Page ◽  
Conceptual Translation

The frizzled (fz) gene of Drosophila is essential for the development of normal tissue polarity in the adult cuticle of Drosophila. In fz mutants the parallel array of hairs and bristles that decorate the cuticle is disrupted. Previous studies have shown that fz encodes a membrane protein with seven putative transmembrane domains, and that it has a complex role in the development of tissue polarity, as there exist both cell-autonomous and cell nonautonomous alleles. We have now examined a larger number of alleles and found that 15 of 19 alleles display cell nonautonomy. We have examined these and other alleles by Western blot analysis and found that most fz mutations result in altered amounts of Fz protein, and many also result in a Fz protein that migrates aberrantly in SDS-PAGE. We have sequenced a subset of these alleles. Cell nonautonomous fz alleles were found to be associated with mutations that altered amino acids in all regions of the Fz protein. Notably, the four cell-autonomous mutations were all in a proline residue located in the presumptive first cytoplasmic loop of the protein. We have also cloned and sequenced the fz gene from D. virilis. Conceptual translation of the D. virilis open reading frame indicates that the Fz protein is unusually well conserved. Indeed, in the putative cytoplasmic domains the Fz proteins of the two species are identical.

Single Amino Acid Based Self-Assemblies of Cysteine and Methionine

2018 ◽  
Author(s):  
Nidhi Gour ◽  
Bharti Koshti ◽  
Chandra Kanth P. ◽  
Dhruvi Shah ◽  
Vivek Shinh Kshatriya ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Self Assembly ◽  
Aromatic Amino Acids ◽  
Neutral Condition ◽  
Single Amino Acid ◽  
Binding Assays ◽  
Human Neuroblastoma ◽  
Cytotoxicity Assays ◽  
First Time

We report for the very first time self-assembly of Cysteine and Methionine to discrenible strucutres under neutral condition. To get insights into the structure formation, thioflavin T and Congo red binding assays were done which revealed that aggregates may not have amyloid like characteristics. The nature of interactions which lead to such self-assemblies was purported by coincubating assemblies in urea and mercaptoethanol. Further interaction of aggregates with short amyloidogenic dipeptide diphenylalanine (FF) was assessed. While cysteine aggregates completely disrupted FF fibres, methionine albeit triggered fibrillation. The cytotoxicity assays of cysteine and methionine structures were performed on Human Neuroblastoma IMR-32 cells which suggested that aggregates are not cytotoxic in nature and thus, may not have amyloid like etiology. The results presented in the manuscript are striking, since to the best of our knowledge,this is the first report which demonstrates that even non-aromatic amino acids (cysteine and methionine) can undergo spontaneous self-assembly to form ordered aggregates.

scholarly journals Uncovering dynamic evolution in the plastid genome of seven Ligusticum species provides insights into species discrimination and phylogenetic implications

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Can Yuan ◽  
Xiufen Sha ◽  
Miao Xiong ◽  
Wenjuan Zhong ◽  
Yu Wei ◽  
...  
Keyword(s):  
Plastid Genome ◽  
Species Discrimination ◽  
Chloroplast Genomes ◽  
Genomic Arrangement ◽  
Phylogenetic Implications ◽  
Solid Foundation ◽  
Conserved Gene ◽  
Fine Resolution ◽  
Genome Scale ◽  
First Time

AbstractLigusticum L., one of the largest members in Apiaceae, encompasses medicinally important plants, the taxonomic statuses of which have been proved to be difficult to resolve. In the current study, the complete chloroplast genomes of seven crucial plants of the best-known herbs in Ligusticum were presented. The seven genomes ranged from 148,275 to 148,564 bp in length with a highly conserved gene content, gene order and genomic arrangement. A shared dramatic decrease in genome size resulted from a lineage-specific inverted repeat (IR) contraction, which could potentially be a promising diagnostic character for taxonomic investigation of Ligusticum, was discovered, without affecting the synonymous rate. Although a higher variability was uncovered in hotspot divergence regions that were unevenly distributed across the chloroplast genome, a concatenated strategy for rapid species identification was proposed because separate fragments inadequately provided variation for fine resolution. Phylogenetic inference using plastid genome-scale data produced a concordant topology receiving a robust support value, which revealed that L. chuanxiong had a closer relationship with L. jeholense than L. sinense, and L. sinense cv. Fuxiong had a closer relationship to L. sinense than L. chuanxiong, for the first time. Our results not only furnish concrete evidence for clarifying Ligusticum taxonomy but also provide a solid foundation for further pharmaphylogenetic investigation.

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