Genes

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Open AccessCommunication

RNAi-Mediated Gene Silencing in a Gonad Organ Culture to Study Sex Determination Mechanisms in Sea Turtle

by Itzel Sifuentes-Romero, Horacio Merchant-Larios, Sarah L. Milton, Norma Moreno-Mendoza, Verónica Díaz-Hernández and Alejandra García-Gasca

Genes 2013, 4(2), 293-305; https://doi.org/10.3390/genes4020293 - 7 Jun 2013

Cited by 16 | Viewed by 8562

Abstract

The autosomal Sry-related gene, Sox9, encodes a transcription factor, which performs an important role in testis differentiation in mammals. In several reptiles, Sox9 is differentially expressed in gonads, showing a significant upregulation during the thermo-sensitive period (TSP) at the male-promoting temperature, [...] Read more.

The autosomal Sry-related gene, Sox9, encodes a transcription factor, which performs an important role in testis differentiation in mammals. In several reptiles, Sox9 is differentially expressed in gonads, showing a significant upregulation during the thermo-sensitive period (TSP) at the male-promoting temperature, consistent with the idea that SOX9 plays a central role in the male pathway. However, in spite of numerous studies, it remains unclear how SOX9 functions during this event. In the present work, we developed an RNAi-based method for silencing Sox9 in an in vitro gonad culture system for the sea turtle, Lepidochelys olivacea. Gonads were dissected as soon as the embryos entered the TSP and were maintained in organ culture. Transfection of siRNA resulted in the decrease of both Sox9 mRNA and protein. Furthermore, we found coordinated expression patterns for Sox9 and the anti-Müllerian hormone gene, Amh, suggesting that SOX9 could directly or indirectly regulate Amh expression, as it occurs in mammals. These results demonstrate an in vitro method to knockdown endogenous genes in gonads from a sea turtle, which represents a novel approach to investigate the roles of important genes involved in sex determination or differentiation pathways in species with temperature-dependent sex determination. Full article

(This article belongs to the Special Issue Gene Silencing)

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Open AccessArticle

Altered Ca²⁺ Homeostasis and Endoplasmic Reticulum Stress in Myotonic Dystrophy Type 1 Muscle Cells

by Annalisa Botta, Adriana Malena, Emanuele Loro, Giulia Del Moro, Matteo Suman, Boris Pantic, Gyorgy Szabadkai and Lodovica Vergani

Genes 2013, 4(2), 275-292; https://doi.org/10.3390/genes4020275 - 4 Jun 2013

Cited by 30 | Viewed by 8011

Abstract

The pathogenesis of Myotonic Dystrophy type 1 (DM1) is linked to unstable CTG repeats in the DMPK gene which induce the mis-splicing to fetal/neonatal isoforms of many transcripts, including those involved in cellular Ca²⁺ homeostasis. Here we monitored the splicing of three [...] Read more.

The pathogenesis of Myotonic Dystrophy type 1 (DM1) is linked to unstable CTG repeats in the DMPK gene which induce the mis-splicing to fetal/neonatal isoforms of many transcripts, including those involved in cellular Ca²⁺ homeostasis. Here we monitored the splicing of three genes encoding for Ca²⁺ transporters and channels (RyR1, SERCA1 and CACN1S) during maturation of primary DM1 muscle cells in parallel with the functionality of the Excitation-Contraction (EC) coupling machinery. At 15 days of differentiation, fetal isoforms of SERCA1 and CACN1S mRNA were significantly higher in DM1 myotubes compared to controls. Parallel functional studies showed that the cytosolic Ca²⁺ response to depolarization in DM1 myotubes did not increase during the progression of differentiation, in contrast to control myotubes. While we observed no differences in the size of intracellular Ca²⁺ stores, DM1 myotubes showed significantly reduced RyR1 protein levels, uncoupling between the segregated ER/SR Ca²⁺ store and the voltage-induced Ca²⁺ release machinery, parallel with induction of endoplasmic reticulum (ER) stress markers. In conclusion, our data suggest that perturbed Ca²⁺ homeostasis, via activation of ER stress, contributes to muscle degeneration in DM1 muscle cells likely representing a premature senescence phenotype. Full article

(This article belongs to the Special Issue Microsatellite Instability)

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Open AccessArticle

Molecular Expression of the Scribble Complex Genes, Dlg, Scrib and Lgl, in Silkworm, Bombyx mori

by Hai-Sheng Qi, Shu-Min Liu, Sheng Li and Zhao-Jun Wei

Genes 2013, 4(2), 264-274; https://doi.org/10.3390/genes4020264 - 30 May 2013

Cited by 2 | Viewed by 6535

Abstract

The Scribble protein complex genes, consisting of lethal giant larvae (Lgl), discs large (Dlg) and scribble (Scrib) genes, are components of an evolutionarily conserved genetic pathway that links the cell polarity in cells of humans and Drosophila. [...] Read more.

The Scribble protein complex genes, consisting of lethal giant larvae (Lgl), discs large (Dlg) and scribble (Scrib) genes, are components of an evolutionarily conserved genetic pathway that links the cell polarity in cells of humans and Drosophila. The tissue expression and developmental changes of the Scribble protein complex genes were documented using qRT-RCR method. The Lgl and Scrib genes could be detected in all the experimental tissues, including fat body, midgut, testis/ovary, wingdisc, trachea, malpighian tubule, hemolymph, prothoracic gland and silk gland. The Dlg gene, mainly expressed only in testis/ovary, could not be detected in prothoracic gland and hemolymph. In fat body, there were two higher expression stages of the three genes. The highest peak of the expression of the Lgl and Scrib genes in wingdisc lay at the 1st day of the 5th instar, but the Dlg gene was at 3rd day of 5th instar. The above results indicate that Scribble complex genes are involved in the process of molting and development of the wingdisc in the silkworm. This will be useful in the future for the elucidation of the detailed biological function of the three genes Scrib, Dlg and Lgl in B. mori. Full article

(This article belongs to the Section Population and Evolutionary Genetics and Genomics)

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Open AccessReview

MicroRNAs and Molecular Mechanisms of Neurodegeneration

by Ilaria Bicchi, Francesco Morena, Simona Montesano, Mario Polidoro and Sabata Martino

Genes 2013, 4(2), 244-263; https://doi.org/10.3390/genes4020244 - 29 May 2013

Cited by 24 | Viewed by 10879

Abstract

During the last few years microRNAs (miRNAs) have emerged as key mediators of post-transcriptional and epigenetic regulation of gene expression. MiRNAs targets, identified through gene expression profiling and studies in animal models, depict a scenario where miRNAs are fine-tuning metabolic pathways and genetic [...] Read more.

During the last few years microRNAs (miRNAs) have emerged as key mediators of post-transcriptional and epigenetic regulation of gene expression. MiRNAs targets, identified through gene expression profiling and studies in animal models, depict a scenario where miRNAs are fine-tuning metabolic pathways and genetic networks in both plants and animals. MiRNAs have shown to be differentially expressed in brain areas and alterations of miRNAs homeostasis have been recently correlated to pathological conditions of the nervous system, such as cancer and neurodegeneration. Here, we review and discuss the most recent insights into the involvement of miRNAs in the neurodegenerative mechanisms and their correlation with significant neurodegenerative disorders. Full article

(This article belongs to the Special Issue RNA Interference)

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Open AccessArticle

High SINE RNA Expression Correlates with Post-Transcriptional Downregulation of BRCA1

by Maureen Peterson, Vicki L. Chandler and Giovanni Bosco

Genes 2013, 4(2), 226-243; https://doi.org/10.3390/genes4020226 - 29 Apr 2013

Cited by 1 | Viewed by 7485

Abstract

Short Interspersed Nuclear Elements (SINEs) are non-autonomous retrotransposons that comprise a large fraction of the human genome. SINEs are demethylated in human disease, but whether SINEs become transcriptionally induced and how the resulting transcripts may affect the expression of protein coding genes is [...] Read more.

Short Interspersed Nuclear Elements (SINEs) are non-autonomous retrotransposons that comprise a large fraction of the human genome. SINEs are demethylated in human disease, but whether SINEs become transcriptionally induced and how the resulting transcripts may affect the expression of protein coding genes is unknown. Here, we show that downregulation of the mRNA of the tumor suppressor gene BRCA1 is associated with increased transcription of SINEs and production of sense and antisense SINE small RNAs. We find that BRCA1 mRNA is post-transcriptionally down-regulated in a Dicer and Drosha dependent manner and that expression of a SINE inverted repeat with sequence identity to a BRCA1 intron is sufficient for downregulation of BRCA1 mRNA. These observations suggest that transcriptional activation of SINEs could contribute to a novel mechanism of RNA mediated post-transcriptional silencing of human genes. Full article

(This article belongs to the Special Issue Gene Silencing)

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Open AccessArticle

Trapping DNA Replication Origins from the Human Genome

by Toshihiko Eki, Yasufumi Murakami and Fumio Hanaoka

Genes 2013, 4(2), 198-225; https://doi.org/10.3390/genes4020198 - 17 Apr 2013

Viewed by 9551

Abstract

Synthesis of chromosomal DNA is initiated from multiple origins of replication in higher eukaryotes; however, little is known about these origins’ structures. We isolated the origin-derived nascent DNAs from a human repair-deficient cell line by blocking the replication forks near the origins using [...] Read more.

Synthesis of chromosomal DNA is initiated from multiple origins of replication in higher eukaryotes; however, little is known about these origins’ structures. We isolated the origin-derived nascent DNAs from a human repair-deficient cell line by blocking the replication forks near the origins using two different origin-trapping methods (i.e., UV- or chemical crosslinker-treatment and cell synchronization in early S phase using DNA replication inhibitors). Single-stranded DNAs (of 0.5–3 kb) that accumulated after such treatments were labeled with bromodeoxyuridine (BrdU). BrdU-labeled DNA was immunopurified after fractionation by alkaline sucrose density gradient centrifugation and cloned by complementary-strand synthesis and PCR amplification. Competitive PCR revealed an increased abundance of DNA derived from known replication origins (c-myc and lamin B2 genes) in the nascent DNA fractions from the UV-treated or crosslinked cells. Nucleotide sequences of 85 and 208 kb were obtained from the two libraries (I and II) prepared from the UV-treated log-phase cells and early S phase arrested cells, respectively. The libraries differed from each other in their G+C composition and replication-related motif contents, suggesting that differences existed between the origin fragments isolated by the two different origin-trapping methods. The replication activities for seven out of 12 putative origin loci from the early-S phase cells were shown by competitive PCR. We mapped 117 (library I) and 172 (library II) putative origin loci to the human genome; approximately 60% and 50% of these loci were assigned to the G-band and intragenic regions, respectively. Analyses of the flanking sequences of the mapped loci suggested that the putative origin loci tended to associate with genes (including conserved sites) and DNase I hypersensitive sites; however, poor correlations were found between such loci and the CpG islands, transcription start sites, and K27-acetylated histone H3 peaks. Full article

(This article belongs to the Special Issue DNA Replication)

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Open AccessReview

Non-Neuronal Functions of the M₂Muscarinic Acetylcholine Receptor

by Wymke Ockenga, Sina Kühne, Simone Bocksberger, Antje Banning and Ritva Tikkanen

Genes 2013, 4(2), 171-197; https://doi.org/10.3390/genes4020171 - 2 Apr 2013

Cited by 22 | Viewed by 27021

Abstract

Acetylcholine is an important neurotransmitter whose effects are mediated by two classes of receptors. The nicotinic acetylcholine receptors are ion channels, whereas the muscarinic receptors belong to the large family of G protein coupled seven transmembrane helix receptors. Beyond its function in neuronal [...] Read more.

Acetylcholine is an important neurotransmitter whose effects are mediated by two classes of receptors. The nicotinic acetylcholine receptors are ion channels, whereas the muscarinic receptors belong to the large family of G protein coupled seven transmembrane helix receptors. Beyond its function in neuronal systems, it has become evident that acetylcholine also plays an important role in non-neuronal cells such as epithelial and immune cells. Furthermore, many cell types in the periphery are capable of synthesizing acetylcholine and express at least some of the receptors. In this review, we summarize the non-neuronal functions of the muscarinic acetylcholine receptors, especially those of the M₂muscarinic receptor in epithelial cells. We will review the mechanisms of signaling by the M₂ receptor but also the cellular trafficking and ARF6 mediated endocytosis of this receptor, which play an important role in the regulation of signaling events. In addition, we provide an overview of the M₂ receptor in human pathological conditions such as autoimmune diseases and cancer. Full article

(This article belongs to the Special Issue Signal Transduction)

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Open AccessReview

Signaling Pathways in Exosomes Biogenesis, Secretion and Fate

by Lorena Urbanelli, Alessandro Magini, Sandra Buratta, Alessandro Brozzi, Krizia Sagini, Alice Polchi, Brunella Tancini and Carla Emiliani

Genes 2013, 4(2), 152-170; https://doi.org/10.3390/genes4020152 - 28 Mar 2013

Cited by 305 | Viewed by 18834

Abstract

Exosomes are small extracellular vesicles (30–100 nm) derived from the endosomal system, which have raised considerable interest in the last decade. Several studies have shown that they mediate cell-to-cell communication in a variety of biological processes. Thus, in addition to cell-to-cell direct interaction [...] Read more.

Exosomes are small extracellular vesicles (30–100 nm) derived from the endosomal system, which have raised considerable interest in the last decade. Several studies have shown that they mediate cell-to-cell communication in a variety of biological processes. Thus, in addition to cell-to-cell direct interaction or secretion of active molecules, they are now considered another class of signal mediators. Exosomes can be secreted by several cell types and retrieved in many body fluids, such as blood, urine, saliva and cerebrospinal fluid. In addition to proteins and lipids, they also contain nucleic acids, namely mRNA and miRNA. These features have prompted extensive research to exploit them as a source of biomarkers for several pathologies, such as cancer and neurodegenerative disorders. In this context, exosomes also appear attractive as gene delivery vehicles. Furthermore, exosome immunomodulatory and regenerative properties are also encouraging their application for further therapeutic purposes. Nevertheless, several issues remain to be addressed: exosome biogenesis and secretion mechanisms have not been clearly understood, and physiological functions, as well as pathological roles, are far from being satisfactorily elucidated. Full article

(This article belongs to the Special Issue Signal Transduction)

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Open AccessArticle

A Novel Function for the Conserved Glutamate Residue in the Walker B Motif of Replication Factor C

by Ankita Chiraniya, Jeff Finkelstein, Mike O'Donnell and Linda B. Bloom

Genes 2013, 4(2), 134-151; https://doi.org/10.3390/genes4020134 - 26 Mar 2013

Cited by 6 | Viewed by 8610

Abstract

In all domains of life, sliding clamps tether DNA polymerases to DNA to increase the processivity of synthesis. Clamp loaders load clamps onto DNA in a multi-step process that requires ATP binding and hydrolysis. Like other AAA+ proteins, clamp loaders contain conserved Walker [...] Read more.

In all domains of life, sliding clamps tether DNA polymerases to DNA to increase the processivity of synthesis. Clamp loaders load clamps onto DNA in a multi-step process that requires ATP binding and hydrolysis. Like other AAA+ proteins, clamp loaders contain conserved Walker A and Walker B sequence motifs, which participate in ATP binding and hydrolysis, respectively. Mutation of the glutamate residue in Walker B motifs (or DExx-boxes) in AAA+ proteins typically reduces ATP hydrolysis by as much as a couple orders of magnitude, but has no effect on ATP binding. Here, the Walker B Glu in each of the four active ATP sites of the eukaryotic clamp loader, RFC, was mutated to Gln and Ala separately, and ATP binding- and hydrolysis-dependent activities of the quadruple mutant clamp loaders were characterized. Fluorescence-based assays were used to measure individual reaction steps required for clamp loading including clamp binding, clamp opening, DNA binding and ATP hydrolysis. Our results show that the Walker B mutations affect ATP-binding-dependent interactions of RFC with the clamp and DNA in addition to reducing ligand-dependent ATP hydrolysis activity. Here, we show that the Walker B glutamate is required for ATP-dependent ligand binding activity, a previously unknown function for this conserved Glu residue in RFC. Full article

(This article belongs to the Special Issue DNA Replication)

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Open AccessReview

The Role of Mitogen-Activated Protein Kinase-Activated Protein Kinases (MAPKAPKs) in Inflammation

by Ugo Moens, Sergiy Kostenko and Baldur Sveinbjørnsson

Genes 2013, 4(2), 101-133; https://doi.org/10.3390/genes4020101 - 26 Mar 2013

Cited by 158 | Viewed by 13805

Abstract

Mitogen-activated protein kinase (MAPK) pathways are implicated in several cellular processes including proliferation, differentiation, apoptosis, cell survival, cell motility, metabolism, stress response and inflammation. MAPK pathways transmit and convert a plethora of extracellular signals by three consecutive phosphorylation events involving a MAPK kinase [...] Read more.

Mitogen-activated protein kinase (MAPK) pathways are implicated in several cellular processes including proliferation, differentiation, apoptosis, cell survival, cell motility, metabolism, stress response and inflammation. MAPK pathways transmit and convert a plethora of extracellular signals by three consecutive phosphorylation events involving a MAPK kinase kinase, a MAPK kinase, and a MAPK. In turn MAPKs phosphorylate substrates, including other protein kinases referred to as MAPK-activated protein kinases (MAPKAPKs). Eleven mammalian MAPKAPKs have been identified: ribosomal-S6-kinases (RSK1-4), mitogen- and stress-activated kinases (MSK1-2), MAPK-interacting kinases (MNK1-2), MAPKAPK-2 (MK2), MAPKAPK-3 (MK3), and MAPKAPK-5 (MK5). The role of these MAPKAPKs in inflammation will be reviewed. Full article

(This article belongs to the Special Issue Signal Transduction)

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Genes, Volume 4, Issue 2 (June 2013) – 10 articles , Pages 101-305

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