Molecular Characterization and Functional Localization of a Novel SUMOylation Gene in Oryza sativa

Small ubiquitin-related modifier (SUMO) regulates the cellular function of diverse proteins through post-translational modifications. The current study defined a new homolog of SUMO genes in the rice genome and named it OsSUMO7. Putative protein analysis of OsSUMO7 detected SUMOylation features, including di-glycine (GG) and consensus motifs (ΨKXE/D) for the SUMOylation site. Phylogenetic analysis demonstrated the high homology of OsSUMO7 with identified rice SUMO genes, which indicates that the OsSUMO7 gene is an evolutionarily conserved SUMO member. RT-PCR analysis revealed that OsSUMO7 was constitutively expressed in all plant organs. Bioinformatic analysis defined the physicochemical properties and structural model prediction of OsSUMO7 proteins. A red fluorescent protein (DsRed), fused with the OsSUMO7 protein, was expressed and localized mainly in the nucleus and formed nuclear subdomain structures. The fusion proteins of SUMO-conjugating enzymes with the OsSUMO7 protein were co-expressed and co-localized in the nucleus and formed nuclear subdomains. This indicated that the OsSUMO7 precursor is processed, activated, and transported to the nucleus through the SUMOylation system of the plant cell.

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Generation of Marker-Free Transgenic Rice Resistant to Rice Blast Disease Using Ac/Ds Transposon-Mediated Transgene Reintegration System

Frontiers in Plant Science ◽

10.3389/fpls.2021.644437 ◽

2021 ◽

Vol 12 ◽

Author(s):

Xin Li ◽

Longyu Pan ◽

Dongling Bi ◽

Xudan Tian ◽

Lihua Li ◽

...

Keyword(s):

Transgenic Rice ◽

Dna Sequences ◽

Rice Blast ◽

Fluorescent Protein ◽

Rice Genome ◽

Rice Blast Disease ◽

Blast Disease ◽

Pcr Analysis ◽

Transgenic Breeding ◽

Marker Free

Rice blast is one of the most serious diseases of rice and a major threat to rice production. Breeding disease-resistant rice is one of the most economical, safe, and effective measures for the control of rice blast. As a complement to traditional crop breeding, the transgenic method can avoid the time-consuming process of crosses and multi-generation selection. In this study, maize (Zea mays) Activator (Ac)/Dissociation (Ds) transposon vectors carrying green fluorescent protein (GFP) and red fluorescent protein (mCherry) genetic markers were used for generating marker-free transgenic rice. Double fluorescent protein-aided counterselection against the presence of T-DNA was performed together with polymerase chain reaction (PCR)-based positive selection for the gene of interest (GOI) to screen marker-free progeny. We cloned an RNAi expression cassette of the rice Pi21 gene that negatively regulates resistance to rice blast as a GOI into the Ds element in the Ac/Ds vector and obtained marker-free T1 rice plants from 13 independent transgenic lines. Marker-free and Ds/GOI-homozygous rice lines were verified by PCR and Southern hybridization analysis to be completely free of transgenic markers and T-DNA sequences. qRT-PCR analysis and rice blast disease inoculation confirmed that the marker-free transgenic rice lines exhibited decreased Pi21 expression levels and increased resistance to rice blast. TAIL-PCR results showed that the Ds (Pi21-RNAi) transgenes in two rice lines were reintegrated in intergenic regions in the rice genome. The Ac/Ds vector with dual fluorescent protein markers offers more reliable screening of marker-free transgenic progeny and can be utilized in the transgenic breeding of rice disease resistance and other agronomic traits.

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LYRM1, a novel gene promotes proliferation and inhibits apoptosis of preadipocytes

Acta Endocrinologica ◽

10.1530/eje-08-0518 ◽

2009 ◽

Vol 160 (2) ◽

pp. 177-184 ◽

Cited By ~ 16

Author(s):

Jie Qiu ◽

Chun-Lin Gao ◽

Min Zhang ◽

Rong-Hua Chen ◽

Xia Chi ◽

...

Keyword(s):

Adipose Tissue ◽

Western Blot ◽

Blot Analysis ◽

Western Blot Analysis ◽

Fluorescent Protein ◽

Pcr Analysis ◽

Rt Pcr ◽

Omental Adipose Tissue ◽

Novel Gene ◽

Obese Subjects

ObjectiveTo characterize a novel gene, Homo sapiens LYR motif containing 1 (LYRM1), that is highly expressed in omental adipose tissue of obese subjects.Methods and resultsRT-PCR and western blot analysis confirmed that both mRNA and protein levels of LYRM1 were higher in omental adipose tissue of obese subjects than in normal weight subjects. RT-PCR analysis demonstrated that LYRM1 expression is widely distributed, with the highest levels of expression occurring in adipose tissue. A fusion protein of LYRM1 and green fluorescent protein as well as western blot analysis were used to identify the subcellular localization of LYRM1 in the nucleus. Based on Oil red O staining and the expression profile of specific differentiation markers, ectopic LYRM1 expression was not found to significantly affect adipogenesis. MTT assays and cell cycle analysis showed that LYRM1 promotes preadipocyte proliferation, and data from annexin V-FITC and caspase-3 activity assays further determined that LYRM1 can inhibit apoptosis of preadipocytes.ConclusionsBy increasing cell proliferation and lowering the rate of apoptosis, LYRM1 has the potential to modulate the size of the preadipocyte pool and influence adipose tissue homeostasis.

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Renal principal cell-specific expression of green fluorescent protein in transgenic mice

AJP Renal Physiology ◽

10.1152/ajprenal.0224.2001 ◽

2002 ◽

Vol 283 (6) ◽

pp. F1351-F1364 ◽

Cited By ~ 11

Author(s):

Ludmilla Zharkikh ◽

Xiaohong Zhu ◽

Peter K. Stricklett ◽

Donald E. Kohan ◽

Greg Chipman ◽

...

Keyword(s):

Gene Expression ◽

Green Fluorescent Protein ◽

Transgenic Mice ◽

Fluorescent Protein ◽

Pcr Analysis ◽

Intercalated Cells ◽

Rt Pcr ◽

Specific Expression ◽

Principal Cells ◽

Green Fluorescent

The purpose of this study is to develop transgenic mice with principal cell-specific expression of green fluorescent protein (GFP). After the cloning and sequencing of the mouse aquaporin-2 (AQP2) gene, 9.5 kb of the promoter were used to drive expression of GFP in transgenic mice. In transgenic mice, GFP was selectively expressed in principal cells of the renal collecting duct and not in intercalated cells. Expression was increased by dehydration of mice. AQP2 and GFP expression was maintained in primary cultures of renal medulla that were stimulated with cAMP or vasopressin analogs. GFP-expressing cells were then isolated by fluorescence-activated cell sorting. RT-PCR analysis showed expression of AQP2, AQP3, AQP4, vasopressin type 2 receptor, and cAMP response element binding protein but not H+-ATPase B1 subunit or anion exchanger 1. After expansion of these cells in culture, RT-PCR analysis showed continued expression of the same genes. This pattern of gene expression is that of principal cells rather than intercalated cells. This transgenic mouse model can be used in future studies of gene expression during the development, differentiation, and maturation of renal principal cells.

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Cloning of the cDNA Encoding Human Platelet CD36: Comparison to PCR Amplified Fragments of Monocyte, Endothelial and HEL Cells

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649613 ◽

1993 ◽

Vol 70 (03) ◽

pp. 500-505 ◽

Cited By ~ 17

Author(s):

B Wyler ◽

L Daviet ◽

H Bortkiewicz ◽

J-C Bordet ◽

J L McGregor

Keyword(s):

Apparent Molecular Weight ◽

Platelet Glycoprotein ◽

Rt Pcr ◽

Post Translational Modifications ◽

Hel Cells ◽

Flanking Region ◽

Polymerase Chain ◽

A Minor ◽

Flanking Regions ◽

Cdna Fragments

SummaryGlycoprotein CD36, also known as GPIIIb or GPIV, is a major platelet glycoprotein that bears the newly identified Naka alloantigen. The aim of this study was to clone platelet CD36 and investigate other forms of CD36-cDNA present in monocytes, endothelial and HEL cells. RNA from above mentioned cells were reverse transcribed (RT), using specific primers for CD36, and amplified by the polymerase chain reaction (PCR) technique. Sequencing the different amplified platelet derived cDNA fragments, spanning the whole coding and flanking regions, showed the near identity between platelet and CD36-placenta cDNA. Platelet CD36-cDNA cross-hybridized, in Southern blots, with RT-PCR amplified cDNA originating from monocytes, endothelial and HEL cells. However, monocytes showed a RT-PCR amplified cDNA fragment (561 bp) that was present in platelets and placenta but not on endothelial on HEL-cells. Northern blot analysis of platelet RNA hybridized with placenta CD36 indicated the presence of a major (1.95 kb) and a minor (0.95 kb) transcript. The 1.95 kb transcript was the only one observed on Northern blots of monocytes, endothelial and HEL cells. These results indicate that the structure of CD36 expressed in platelets is similar, with the exception of the 3’ flanking region, to that of placenta. Differences in apparent molecular weight between CD36 and CD36-like glycoproteins may be due to post-translational modifications.

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Phenotypical and Functional Characteristics of Human Regulatory T Cells during Ex Vivo Maturation from CD4+ T Lymphocytes

Applied Sciences ◽

10.3390/app11135776 ◽

2021 ◽

Vol 11 (13) ◽

pp. 5776

Author(s):

Varvara G. Blinova ◽

Natalia S. Novachly ◽

Sofya N. Gippius ◽

Abdullah Hilal ◽

Yulia A. Gladilina ◽

...

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Ex Vivo ◽

Pcr Analysis ◽

Mrna Levels ◽

Rt Pcr ◽

Inflammatory Reactions ◽

Effector Cells ◽

Cycle Regulation ◽

Further Development

Regulatory T cells (Tregs) participate in the negative regulation of inflammatory reactions by suppressing effector cells. In a number of autoimmune disorders, the suppressive function and/or the number of Tregs is compromised. The lack of active functioning Tregs can be restored with adoptive transfer of expanded ex vivo autologous Tregs. In our study, we traced the differentiation and maturation of Tregs CD4+CD25+FoxP3+CD127low over 7 days of cultivation from initial CD4+ T cells under ex vivo conditions. The resulting ex vivo expanded cell population (eTregs) demonstrated the immune profile of Tregs with an increased capacity to suppress the proliferation of target effector cells. The expression of the FoxP3 gene was upregulated within the time of expansion and was associated with gradual demethylation in the promotor region of the T cell-specific demethylation region. Real-time RT-PCR analysis revealed changes in the expression profile of genes involved in cell cycle regulation. In addition to FOXP3, the cells displayed elevated mRNA levels of Ikaros zinc finger transcription factors and the main telomerase catalytic subunit hTERT. Alternative splicing of FoxP3, hTERT and IKZF family members was demonstrated to be involved in eTreg maturation. Our data indicate that expanded ex vivo eTregs develop a Treg-specific phenotype and functional suppressive activity. We suggest that eTregs are not just expanded but transformed cells with enhanced capacities of immune suppression. Our findings may influence further development of cell immunosuppressive therapy based on regulatory T cells.

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Role of Innate Inflammation in the Regulation of Tissue Remodeling during Tooth Eruption

Dentistry Journal ◽

10.3390/dj9010007 ◽

2021 ◽

Vol 9 (1) ◽

pp. 7

Author(s):

Yusuke Makino ◽

Kaoru Fujikawa ◽

Miwako Matsuki-Fukushima ◽

Satoshi Inoue ◽

Masanori Nakamura

Keyword(s):

Bacterial Infections ◽

Tooth Movement ◽

Tooth Eruption ◽

Intercellular Adhesion Molecule ◽

Enamel Organ ◽

Pcr Analysis ◽

Maturation Stage ◽

Oral Epithelium ◽

Rt Pcr ◽

Inflammatory Reactions

Tooth eruption is characterized by a coordinated complex cascade of cellular and molecular events that promote tooth movement through the eruptive pathway. During tooth eruption, the stratum intermedium structurally changes to the papillary layer with tooth organ development. We previously reported intercellular adhesion molecule-1 (ICAM-1) expression on the papillary layer, which is the origin of the ICAM-1-positive junctional epithelium. ICAM-1 expression is induced by proinflammatory cytokines, including interleukin-1 and tumor necrosis factor. Inflammatory reactions induce tissue degradation. Therefore, this study aimed to examine whether inflammatory reactions are involved in tooth eruption. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed sequential expression of hypoxia-induced factor-1α, interleukin-1β, and chemotactic factors, including keratinocyte-derived chemokine (KC) and macrophage inflammatory protein-2 (MIP-2), during tooth eruption. Consistent with the RT-PCR results, immunohistochemical analysis revealed KC and MIP-2 expression in the papillary layer cells of the enamel organ from the ameloblast maturation stage. Moreover, there was massive macrophage and neutrophil infiltration in the connective tissue between the tooth organ and oral epithelium during tooth eruption. These findings suggest that inflammatory reactions might be involved in the degradation of tissue overlying the tooth organ. Further, these reactions might be induced by hypoxia in the tissue overlying the tooth organ, which results from decreased capillaries in the tissue. Our findings indicate that bacterial infections are not associated with the eruption process. Therefore, tooth eruption might be regulated by innate inflammatory mechanisms.

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RT-PCR analysis of Deformed wing virus in honeybees (Apis mellifera) and mites (Varroa destructor)

Journal of General Virology ◽

10.1099/vir.0.81401-0 ◽

2005 ◽

Vol 86 (12) ◽

pp. 3419-3424 ◽

Cited By ~ 210

Author(s):

Constanze Yue ◽

Elke Genersch

Keyword(s):

Varroa Destructor ◽

Pcr Analysis ◽

Body Parts ◽

Rt Pcr ◽

Larval Food ◽

Viral Pathogen ◽

Deformed Wing Virus ◽

Significant Difference ◽

Almost All ◽

Viral Sequences

Deformed wing virus (DWV) is a honeybee viral pathogen either persisting as an inapparent infection or resulting in wing deformity. The occurrence of deformity is associated with the transmission of DWV through Varroa destructor during pupal stages. Such infections with DWV add to the pathology of V. destructor and play a major role in colony collapse in the course of varroosis. Using a recently developed RT-PCR protocol for the detection of DWV, individual bees and mites originating from hives differing in Varroa infestation levels and the occurrence of crippled bees were analysed. It was found that 100 % of both crippled and asymptomatic bees were positive for DWV. However, a significant difference in the spatial distribution of DWV between asymptomatic and crippled bees could be demonstrated: when analysing head, thorax and abdomen of crippled bees, all body parts were always strongly positive for viral sequences. In contrast, for asymptomatic bees viral sequences could be detected in RNA extracted from the thorax and/or abdomen but never in RNA extracted from the head. DWV replication was demonstrated in almost all DWV-positive body parts of infected bees. Analysing individual mites for the presence of DWV revealed that the percentage of DWV-positive mites differed between mite populations. In addition, it was demonstrated that DWV was able to replicate in some but not all mites. Interestingly, virus replication in mites was correlated with wing deformity. DWV was also detected in the larval food, implicating that in addition to transmission by V. destructor DWV is also transmitted by feeding.

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Reproductive failure in mice lacking inter-alpha-trypsin inhibitor (ITI) – ITI target genes in mouse ovary identified by microarray analysis

Journal of Endocrinology ◽

10.1677/joe.1.05803 ◽

2004 ◽

Vol 183 (1) ◽

pp. 29-38 ◽

Cited By ~ 14

Author(s):

Mika Suzuki ◽

Hiroshi Kobayashi ◽

Yoshiko Tanaka ◽

Naohiro Kanayama ◽

Toshihiko Terao

Keyword(s):

Real Time ◽

Trypsin Inhibitor ◽

Target Genes ◽

Reproductive Function ◽

Pcr Analysis ◽

Rt Pcr ◽

Reproductive Process ◽

Female Mice ◽

Retinoid Metabolism ◽

Regulatory Effects

Bikunin, a Kunitz-type protease inhibitor, is found in blood and urine. It has been established by two laboratories independently that the bikunin knockout female mice display a severe reduction in fertility: the cumulus oophorus has a defect in forming the extracellular hyaluronan-rich matrix during expansion. Proteins of the inter-alpha-trypsin inhibitor (ITI) family are eliminated in mice in which the bikunin gene has been inactivated, since bikunin is essential for their biosynthesis. Proteins of the ITI family may contribute to the microenvironment in which ovulation takes place. It is not clear, however, whether a single mechanism affects the reproductive function including ovulation. For identifying the full repertoire of the ITI deficiency-related genes, a cDNA microarray hybridization screening was conducted using mRNA from ovaries of wild-type or bik−/− female mice. A number of genes were identified and their regulation was confirmed by real-time RT-PCR analysis. Our screen identified that 29 (0.7%) and 5 genes (0.1%) of the genes assayed were, respectively, up- and down-regulated twofold or more. The identified genes can be classified into distinct subsets. These include stress-related, apoptosis-related, proteases, signaling molecules, aging-related, cytokines, hyaluronan metabolism and signaling, reactive oxygen species-related, and retinoid metabolism, which have previously been implicated in enhancing follicle development and/or ovulation. Real-time RT-PCR analysis confirmed that these genes were up- and down-regulated two- to tenfold by bikunin knockout. These studies demonstrate that proteins of the ITI family may exert potent regulatory effects on a major physiological reproductive process, ovulation.

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