scholarly journals Alternative splicing of ClC-6 (a member of the CIC chloride-channel family) transcripts generates three truncated isoforms one of which, ClC-6c, is kidney-specific

1997 ◽  
Vol 325 (1) ◽  
pp. 269-276 ◽  
Author(s):  
Jan EGGERMONT ◽  
Gunnar BUYSE ◽  
Thomas VOETS ◽  
Jan TYTGAT ◽  
Humbert DE SMEDT ◽  
...  
Keyword(s):  
Amino Acids ◽  
Alternative Splicing ◽  
Chloride Channels ◽  
K562 Cells ◽  
Tissue Expression ◽  
Protein Isoforms ◽  
Mouse Tissue ◽  
Pcr Analysis ◽  
Reverse Transcription Pcr ◽  
Hydropathy Plot

ClC-6 is a protein that structurally belongs to the family of ClC-type chloride channels. We now report the identification of three additional ClC-6 isoforms that are truncated because of alternative splicing. We have isolated, from human K562 cells, four types of ClC-6 cDNAs that encode four distinct ClC-6 protein isoforms. ClC-6a (869 amino acids) corresponds to the previously published ClC-6 protein [Brandt and Jentsch (1995) FEBS Lett. 377, 15–20] and it has a canonical ClC structure. However, ClC-6b (320 amino acids), ClC-6c (353 amino acids) and ClC-6d (308 amino acids) are truncated at their C-termini. Hydropathy-plot analysis indicates that the shortened isoforms contain maximally four (ClC-6b and -6d) or seven (ClC-6c) transmembrane domains. Sequence analysis of a human genomic ClC-6 fragment indicates that the cDNA variability arises from alternative splicing at two different positions: the first alternative site consists of an intron flanked by two alternative donor sites and two alternative acceptor sites, the second being due to an exon that is optionally included or excluded. Reverse-transcription-PCR analysis of ClC-6 expression in human cell lines and tissues shows that the majority (83%) of ClC-6 mRNAs consists of ClC-6a or ClC-6c messengers. Furthermore, in a mouse tissue panel, the ClC-6a mRNA has a relatively broad tissue expression pattern, since it could be detected in brain, kidney, testis, skeletal muscle, thymus and pancreas. In contrast, expression of ClC-6c is more restricted, since it was only detected in kidney.

Differential Modulation of the β-Like Globin Genes by KLFs Isolated with a γ-Globin CACCC Bait.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3637-3637
Author(s):  
Paolo Moi ◽  
Loredana Porcu ◽  
Maria G. Marini ◽  
Isadora Asunis ◽  
Maria G. Loi ◽  
...  
Keyword(s):  
Cell Lines ◽  
Globin Gene ◽  
K562 Cells ◽  
Tissue Expression ◽  
Pcr Analysis ◽  
Globin Genes ◽  
Band Shift ◽  
Hemoglobin Switching ◽  
Specific Complex ◽  
Globin Promoter

Abstract The globin CACCC boxes are absolutely required for the appropriate regulation of the β-like globin genes. While the β-globin CACCC box binds EKLF/KLF1, a likely adult switching factor, analogous factors, interacting with the γ-globin gene and predicted to regulate the fetal stage of hemoglobin switching, have so far been elusive. By using yeast one hybrid assay, we have isolated four KLFs, KLF1, 2, 4, and 6, that bound the γ-CACCC bait. To establish their role in globin regulation and in the switching of hemoglobins, these factors were compared to four other KLFs already established or putative globin regulators, KLF3, 11, 13 and 16, mainly evaluating their ability to bind and transactivate the ε-, γ- and β-globin gene. γ-CACCC binding at variable intensities was confirmed in band shift assay for all four isolated KLFs, for KLF3 and, faintly, for KLF13. The ε- and β-CACCC were bound by the same factors with similar affinities with the exception of KLF3 and KLF13 that bound stronger to the β- and ε- than to the γ-CACCC box. On the other hand, KLF11 and 16 did not produce any specific complex in band shift assays with anyone of the globin CACCC boxes. More relevant differences were observed among the factors in the transactivation of single and dual luciferase reporters in both K562 and MEL cells. In these assays, most factors presented peculiar modulatory properties and specific promoter tropism. Several factors presented bidirectional activity displaying in the same time the capacity to stimulate and repress different globin promoters. KLF1 and 4 were the strongest stimulators of the β-globin promoter in both cell lines, whereas KLF2 activated the β-promoter only in K562 cells. KLF1 and especially KLF4 consistently repressed ε-globin expression especially in MEL cells. KLF3 behaved always as a general globin repressor in MEL cells, but acted as a weak stimulator of the γ- and ε-promoter in K562 cells. KLF4 was the strongest inhibitor of the ε-globin gene. KLF13 significantly stimulated the γ-promoter in both cell lines, whereas KLF3, 4 and 6 showed statistically significant stimulation only in MEL cells. By RT-PCR analysis we found that KLFs were highly variable in their tissue expression and that KLF1, 3 and 13 had the highest expression in erythroid tissues. Thus the level of tissue expression should ultimately determine which factors are really active in physiological conditions. Taken together our binding and expression studies suggest that several KLFs have the potential to modulate the activity of the globin genes and that the resulting globin expression will depend on the vectorial sum of the relative activities of the factors expressed at any given time of development. Furthermore, as some KLFs, like KLF1 and 4, exert opposite effects on fetal and adult globin genes, their role in hemoglobin switching may be direct and not only dependent on their ability to mediate promoter competition for the LCR.

An alternative splice variant of the mouse TRH receptor mRNA is the major form expressed in the mouse pituitary gland

1996 ◽  
Vol 16 (2) ◽  
pp. 197-204 ◽  
Author(s):  
K E Jones ◽  
J H Brubaker ◽  
W W Chin
Keyword(s):  
Amino Acids ◽  
Stop Codon ◽  
Specific Pattern ◽  
Pcr Analysis ◽  
Reverse Transcription Pcr ◽  
Mouse Sequence ◽  
Mouse Cdna ◽  
Rat Pituitary ◽  
Mouse Pituitary ◽  
Species Specific

ABSTRACT The sequences of the mouse and rat TRH receptors (TRH-Rs) show 94% similarity at the protein level. However, they differ significantly at their carboxy terminals, i.e. the mouse TRH-R ends with an asparagine at position 393 while, in the rat, residue 393 is lysine and an additional 19 amino acids are added before the first stop codon. In the mouse cDNA, the sequence encoding these additional amino acids is located 224 bp downstream in the 3′ untranslated region (3′UT). As the mouse TRH-R was cloned from thyrotrope-derived TtT97 tissue and the rat TRH-R from lactotrope-derived GH cell lines, we have investigated whether this difference at the carboxyterminus represents a species-specific or cell type-specific pattern of TRH-R expression. Total RNA was isolated from mouse pituitary and TtT97 tissue, and rat pituitary and GH3 cells. Reverse transcription PCR analysis was performed using primers that would generate DNA fragments including the stop codon in either the mouse or the rat TRH-R and, in the mouse form, the extra 224 bp of 3′UT. This would generate a product of 234 bp from the rat sequence and 441 bp from the mouse sequence. In rat pituitary and GH3 cDNA, PCR generated the expected 234 bp product but not a band representing the mouse sequence. In both mouse pituitary and TtT97 cDNA, neither the expected 441 bp nor the 234 bp fragments were amplified; instead a larger, 829 bp, product was generated. Sequence analysis revealed a 388 bp insertion at position 1663 in the 3′UT compared with the published mouse TRH-R sequence. Ribonuclease protection analysis using this 829 bp fragment as a probe showed that this sequence represented the major TRH-R mRNA species in mouse pituitary and TtT97 RNA. A genomic clone containing this region of the mouse TRH-R gene was isolated and analysis of the sequence in this region revealed that this longer form of the mouse TRH-R could be generated by alternative splicing. In summary, we have shown that the carboxyterminal differences between the mouse and rat TRH-Rs are species-specific rather than cell typespecific, and that the major TRH-R mRNA expressed in mouse pituitary contains an additional 388 bp of 3′UT compared with the published sequence. As a region in the 3′UT of the published mTRH-R sequence has been shown to be important for stability of this mRNA, this additional 3′UT sequence could have major effects on the regulation and stability of the mouse TRH-R mRNA.

scholarly journals MicroRNA-182-5p relieves murine allergic rhinitis via TLR4/NF-κB pathway

Open Medicine ◽  
2020 ◽  
Vol 15 (1) ◽  
pp. 1202-1212
Author(s):  
Aichun Zhang ◽  
Yangzi Jin
Keyword(s):  
Allergic Rhinitis ◽  
Inflammatory Cells ◽  
Lavage Fluid ◽  
Specific Ige ◽  
Inflammatory Factors ◽  
Pcr Analysis ◽  
Reverse Transcription Pcr ◽  
Pathway Activation ◽  
Nasal Lavage Fluid

AbstractAllergic rhinitis (AR) is one of the most common chronic diseases. This study examined whether microRNA (miR)-182-5p plays a role in AR by regulating toll-like receptor 4 (TLR4). First, data demonstrated that TLR4 was a target of miR-182-5p. Subsequently, AR mouse model was established to explore the role of miR-182-5p and TLR4 in AR in vivo. Initially, quantitative reverse transcription-PCR (qRT-PCR) analysis indicated that miR-182-5p was downregulated, while TLR4 expression was upregulated in AR mice. Then we found that miR-182-5p mimic reduced the frequency of sneezing and nose rubbing of the AR mice. In addition, miR-182-5p mimic significantly increased ovalbumin (OVA)-specific IgE and leukotriene C4 expression levels in nasal lavage fluid (NLF) and serum of AR mice. miR-182-5p mimic decreased the number of inflammatory cells in NLF of AR mice. It also reduced the levels of inflammatory factors in the serum of AR mice, such as interleukin (IL)-4, IL-5, IL-13, IL-17 and tumor necrosis factor (TNF)-α, while increasing the release of IFN-γ and IL-2. Finally, miR-182-5p mimic inhibited NF-κB signaling pathway activation in AR mice. However, all effects of miR-182-5p mimic on AR mice were reversed by TLR4-plasmid. In conclusion, miR-182-5p/TLR4 axis may represent a novel therapeutic target for AR.

scholarly journals Exploration of Alternative Splicing Events in Mesenchymal Stem Cells from Human Induced Pluripotent Stem Cells

Genes ◽  
2021 ◽  
Vol 12 (5) ◽  
pp. 737
Author(s):  
Ji-Eun Jeong ◽  
Binna Seol ◽  
Han-Seop Kim ◽  
Jae-Yun Kim ◽  
Yee-Sook Cho
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Alternative Splicing ◽  
Functional Enrichment ◽  
Actin Binding ◽  
Pcr Analysis ◽  
Valuable Insight ◽  
Sequencing Analysis ◽  
Induced Pluripotent ◽  
Insight Into

Although comparative genome-wide transcriptomic analysis has provided insight into the biology of human induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs), the distinct alternative splicing (AS) signatures of iMSCs remain elusive. Here, we performed Illumina RNA sequencing analysis to characterize AS events in iMSCs compared with tissue-derived MSCs. A total of 4586 differentially expressed genes (|FC| > 2) were identified between iMSCs and umbilical cord blood-derived MSCs (UCB-MSCs), including 2169 upregulated and 2417 downregulated genes. Of these, 164 differentially spliced events (BF > 20) in 112 genes were identified between iMSCs and UCB-MSCs. The predominant type of AS found in iMSCs was skipped exons (43.3%), followed by retained introns (19.5%), alternative 3′ (15.2%) and 5′ (12.8%) splice sites, and mutually exclusive exons (9.1%). Functional enrichment analysis showed that the differentially spliced genes (|FC| > 2 and BF > 20) were mainly enriched in functions associated with focal adhesion, extracellular exosomes, extracellular matrix organization, cell adhesion, and actin binding. Splice isoforms of selected genes including TRPT1, CNN2, and AP1G2, identified in sashimi plots, were further validated by RT-PCR analysis. This study provides valuable insight into the biology of iMSCs and the translation of mechanistic understanding of iMSCs into therapeutic applications.

scholarly journals Reverse Transcription-PCR Analysis of Bottled and Natural Mineral Waters for the Presence of Noroviruses

2003 ◽  
Vol 69 (11) ◽  
pp. 6541-6549 ◽  
Author(s):  
Gilbert Thierry Lamothe ◽  
Thierry Putallaz ◽  
Han Joosten ◽  
Joey D. Marugg
Keyword(s):  
Reverse Transcription ◽  
Membrane Filtration ◽  
Limit Of Detection ◽  
Mineral Waters ◽  
Pcr Analysis ◽  
Rna Sequences ◽  
Natural Mineral ◽  
Reverse Transcription Pcr ◽  
Viral Particles ◽  
Nonspecific Amplification

ABSTRACT A seminested reverse transcription-PCR method coupled to membrane filtration was optimized to investigate the presence of norovirus (NV) RNA sequences in bottled and natural mineral waters. The recovery of viral particles by filtration varied between 28 and 45%, while the limit of detection of the overall method ranged from 6 to 95 viral particles. The assay was broadly reactive, as shown by the successful detection of 27 different viral strains representing 12 common genotypes of NVs. A total of 718 bottled and natural mineral water samples were investigated, including 640 samples of finished, spring, and line products (mostly 1 to 1.5 liters), collected from 36 different water brands of various types and from diverse geographic origins over a 2-year period. In addition, 78 samples of larger volume (10 and 400 to 500 liters) and environmental swabs were investigated. From the 1,436 analyses that were performed for the detection of NVs belonging to genogroups I and II, 34 samples (2.44%) were presumptively positive by seminested RT-PCR. However, confirmation by DNA sequence analysis revealed that all presumptive positive results were either due to nonspecific amplification or to cross-contamination. In conclusion, these results do not provide any evidence for the presence of NV genome sequences in bottled waters.

Computation-assisted targeted proteomics of alternative splicing protein isoforms in the human heart

2021 ◽  
Vol 154 ◽  
pp. 92-96
Author(s):  
Yu Han ◽  
Silas D. Wood ◽  
Julianna M. Wright ◽  
Vishantie Dostal ◽  
Edward Lau ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Human Heart ◽  
Protein Isoforms ◽  
Targeted Proteomics

The effects of quercetin on the expression of SREBP-1c mRNA in high-fat diet-induced NAFLD in mice

2021 ◽  
Vol 32 (4) ◽  
pp. 637-644
Author(s):  
Jamal Nasser Saleh Al-maamari ◽  
Mahardian Rahmadi ◽  
Sisca Melani Panggono ◽  
Devita Ardina Prameswari ◽  
Eka Dewi Pratiwi ◽  
...  
Keyword(s):  
High Fat Diet ◽  
Fatty Liver Disease ◽  
Inhibitory Effect ◽  
Pcr Analysis ◽  
Regulator Gene ◽  
High Fat ◽  
Alcoholic Fatty Liver ◽  
Reverse Transcription Pcr ◽  
Hematoxylin Eosin ◽  
Alcoholic Fatty Liver Disease

Abstract Objectives The study aimed to determine the effect of quercetin on the expression of primary regulator gene involved in lipogenesis and triglycerides synthesis in the liver, and the sterol regulatory binding protein-1c (SREBP-1c) mRNA in non-alcoholic fatty liver disease (NAFLD) with a high-fat diet (HFD) model. Methods Fifty-six Balb/c mice were divided into seven groups: standard feed; HFD; HFD and quercetin 50 mg/kg for 28 days; HFD and quercetin 100 mg/kg BW for 28 days; HFD and quercetin 50 mg/kg for 14 days; HFD and quercetin 100 mg/kg for 14 days; HFD and repaired fed for 14 days. Quercetin was administered intraperitoneally. The animals were sacrificed 24 h after the last treatment; the liver was taken for macroscopic, histopathological staining using hematoxylin–eosin and reverse transcription-PCR analysis sample. Results HFD significantly increased the expression of SREBP-1c mRNA; meanwhile, quercetin and repaired feed significantly reduced the expression of SREBP-1c mRNA in the liver. Quercetin at a dose of 50 mg/kg and 100 mg/kg also improved liver cells’ pathological profile in high-fat diet NAFLD. Conclusions The present study suggests that quercetin has an inhibitory effect on SREBP-1c expression and improved liver pathology in NAFLD mice.

scholarly journals Transactivation of Latent Marek's Disease Herpesvirus Genes in QT35, a Quail Fibroblast Cell Line, by Herpesvirus of Turkeys

2000 ◽  
Vol 74 (21) ◽  
pp. 10176-10186 ◽  
Author(s):  
T. Yamaguchi ◽  
S. L. Kaplan ◽  
P. Wakenell ◽  
K. A. Schat
Keyword(s):  
Cell Line ◽  
Fibroblast Cell ◽  
Northern Blotting ◽  
Marek's Disease ◽  
Pcr Analysis ◽  
Marek’S Disease ◽  
Reading Frame ◽  
Reverse Transcription Pcr ◽  
Herpesvirus Of Turkeys ◽  
Serotype 1

ABSTRACT The QT35 cell line was established from a methylcholanthrene-induced tumor in Japanese quail (Coturnix coturnix japonica) (C. Moscovici, M. G. Moscovici, H. Jimenez, M. M. Lai, M. J. Hayman, and P. K. Vogt, Cell 11:95–103, 1977). Two independently maintained sublines of QT35 were found to be positive for Marek's disease virus (MDV)-like genes by Southern blotting and PCR assays. Sequence analysis of fragments of the ICP4, ICP22, ICP27, VP16, meq, pp14, pp38, open reading frame (ORF) L1, and glycoprotein B (gB) genes showed a strong homology with the corresponding fragments of MDV genes. Subsequently, a serotype 1 MDV-like herpesvirus, tentatively name QMDV, was rescued from QT35 cells in chicken kidney cell (CKC) cultures established from 6- to 9-day-old chicks inoculated at 8 days of embryonation with QT35 cells. Transmission electron microscopy failed to show herpesvirus particles in QT35 cells, but typical intranuclear herpesvirus particles were detected in CKCs. Reverse transcription-PCR analysis showed that the following QMDV transcripts were present in QT35 cells: sense and antisense meq, ORF L1, ICP4, and latency-associated transcripts, which are antisense to ICP4. A transcript of approximately 4.5 kb was detected by Northern blotting using total RNA from QT35 cells. Inoculation of QT35 cells with herpesvirus of turkeys (HVT)-infected chicken embryo fibroblasts (CEF) but not with uninfected CEF resulted in the activation of ICP22, ICP27, VP16, pp38, and gB. In addition, the level of ICP4 mRNA was increased compared to that in QT35 cells. The activation by HVT resulted in the production of pp38 protein. It was not possible to detect if the other activated genes were translated due to the lack of serotype 1-specific monoclonal antibodies.

miR-369-3p serves as prognostic factor and regulates cancer progression of hepatocellular carcinoma

2021 ◽  
Author(s):  
Can Chen ◽  
Yi Zong ◽  
Jiaojiao Tang ◽  
Ruisheng Ke ◽  
Lizhi Lv ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Proliferation ◽  
Cancer Progression ◽  
Cell Counting ◽  
Migration And Invasion ◽  
Pcr Analysis ◽  
Reverse Transcription Pcr ◽  
Huh7 Cells

Background: The aim of this study was to investigate the role of miR-369-3p in hepatocellular carcinoma (HCC). Materials & methods: The expression levels of miR-369-3p were detected using the quantitative real-time reverse transcription-PCR analysis. The cell counting kit-8 and transwell assays were used to explore the effects of miR-369-3p on cell proliferation, migration and invasion of HCC cells. Results: The miR-369-3p expression was downregulated in HCC tissues and cell lines, in comparison to the normal controls, respectively. In vitro, overexpression of miR-369-3p in Hep 3B and Huh7 cells inhibited cell proliferation, migration and invasion. SOX4 was a direct target of miR-369-3p. Conclusion: Our results suggested that miR-369-3p may be a tumor suppressor in HCC by targeting SOX4.

scholarly journals Integrative Visual Analysis of the Effects of Alternative Splicing on Protein Domain Interaction Networks

2008 ◽  
Vol 5 (2) ◽  
Author(s):  
Dorothea Emig ◽  
Melissa S. Cline ◽  
Karsten Klein ◽  
Anne Kunert ◽  
Petra Mutzel ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Protein Interactions ◽  
Visual Analysis ◽  
Protein Isoforms ◽  
Interaction Networks ◽  
Protein Domain ◽  
Domain Interaction ◽  
Exon Arrays ◽  
Exon Expression ◽  
Splicing Patterns

SummaryProteins and their interactions are essential for the functioning of all organisms and for understanding biological processes. Alternative splicing is an important molecular mechanism for increasing the protein diversity in eukaryotic cells. Splicing events that alter the protein structure and the domain composition can be responsible for the regulation of protein interactions and the functional diversity of different tissues. Discovering the occurrence of splicing events and studying protein isoforms have become feasible using Affymetrix Exon Arrays. Therefore, we have developed the versatile Cytoscape plugin DomainGraph that allows for the visual analysis of protein domain interaction networks and their integration with exon expression data. Protein domains affected by alternative splicing are highlighted and splicing patterns can be compared.

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