Rad Gene Research Articles

RAD gene family analysis in cotton provides some key genes for flowering and stress tolerance in upland cotton G. hirsutum

BackgroundRADIALIS (RAD), belongs to the MYB gene family and regulates a variety of functions including floral dorsoventral asymmetry in Antirrhinum majus and development of fruit proteins in Solanum lycopersicum. RAD genes contain an SNF2_N superfamily domain. Here, we comprehensively identified 68 RAD genes from six different species including Arabidopsis and five species of cotton.ResultsPhylogenetic analysis classified RAD genes into five groups. Gene structure, protein motifs and conserved amino acid residues indicated that GhRAD genes were highly conserved during the evolutionary process. Chromosomal location information showed that GhRAD genes were distributed unevenly on different chromosomes. Collinearity and selection pressure analysis indicated RAD gene family expansion in G. hirsutum and G. barbadense with purifying selection pressure. Further, various growth and stress related promotor cis-acting elements were observed. Tissue specific expression level indicated that most GhRAD genes were highly expressed in roots and flowers (GhRAD2, GhRAD3, GhRAD4 and GhRAD11). Next, GhRAD genes were regulated by phytohormonal stresses (JA, BL and IAA). Moreover, Ghi-miRN1496, Ghi-miR1440, Ghi-miR2111b, Ghi-miR2950a, Ghi-miR390a, Ghi-miR390b and Ghi-miR7495 were the miRNAs targeting most of GhRAD genes.ConclusionsOur study revealed that RAD genes are evolutionary conserved and might be involved in different developmental processes and hormonal stress response. Data presented in our study could be used as the basis for future studies of RAD genes in cotton.

Life science experiments performed in space in the ISS/Kibo facility and future research plans

Over the past several years, current techniques in molecular biology have been used to perform experiments in space, focusing on the nature and effects of space radiation. In the Japanese ‘Kibo’ facility in the International Space Station (ISS), the Japan Aerospace Exploration Agency (JAXA) has performed five life science experiments since 2009, and two additional experiments are currently in progress. The first life science experiment in space was the ‘Rad Gene’ project, which utilized two human cultured lymphoblastoid cell lines containing a mutated p53 gene (mp53) and a parental wild-type p53 gene (wtp53) respectively. Four parameters were examined: (i) detecting space radiation–induced DSBs by observing γH2AX foci; (ii) observing p53-dependent gene expression during space flight; (iii) observing p53-dependent gene expression after space flight; and (iv) observing the adaptive response in the two cell lines containing the mutated and wild type p53 genes after exposure to space radiation. These observations were completed and have been reported, and this paper is a review of these experiments. In addition, recent new information from space-based experiments involving radiation biology is presented here. These experiments involve human cultured cells, silkworm eggs, mouse embryonic stem cells and mouse eggs in various experiments designed by other principal investigators in the ISS/Kibo. The progress of Japanese science groups involved in these space experiments together with JAXA are also discussed here. The Japanese Society for Biological Sciences in Space (JSBSS), the Utilization Committee of Space Environment Science (UCSES) and the Science Council of Japan (ACJ) have supported these new projects and new experimental facilities in ISS/Kibo. Currently, these organizations are proposing new experiments for the ISS through 2024.

Myosin light chain 3f attenuates age‐induced decline in contractile velocity in MHC type II single muscle fibers

Aging is characterized by a progressive loss of muscle mass and impaired contractility (e.g., decline in force, velocity, and power). Although the slowing of contraction speed in aging muscle is well described, the underlying molecular mechanisms responsible for the decrement in speed are unknown. Myosin heavy chain (MHC) isoforms are the primary molecules determining contractile velocity; however, the contraction speed of single fibers within a given MHC isoform type is variable. Recent evidence proposes that the decline in shortening velocity (Vo) with aging is associated with a decrease in the relative content of essential myosin light chain 3f (MLC(3f) ) isoform. In the current study, we first evaluated the relative content of MLC(3f) isoform and Vo in adult and old rats. We then used recombinant adenovirus (rAd) gene transfer technology to increase MLC(3f) protein content in the MHC type II semimembranosus muscle (SM). We hypothesized that (i) aging would decrease the relative MLC(3f) content and Vo in type II fibers, and (ii) increasing the MLC(3f) content would restore the age-induced decline in Vo. We found that there was an age-related decrement in relative MLC(3f) content and Vo in MHC type II fibers. Increasing MLC(3f) content, as indicated by greater % MLC(3f) and MLC(3f) /MLC(2f) ratio, provided significant protection against age-induced decline in Vo without influencing fiber diameter, force generation, MHC isoform distribution, or causing cellular damage. To the best of our knowledge, these are the first data to demonstrate positive effects of MLC(3f) against slowing of contractile function in aged skeletal muscle.

Increased Myosin Light Chain 3f Content Restores Age‐Induced Slowing of Single Skeletal Muscle Fiber Contraction

Aging is characterized by a progressive loss of muscle mass and impaired contractile functions (e.g. decline in force, velocity and power). Although these phenomena in aging muscle are well described, the underlying molecular mechanisms responsible for a decrease in contraction speed are not defined. Evidence suggests that the slowing of muscle contraction, as demonstrated by slower unloaded shortening velocity (Vo), is correlated with a decrease in relative myosin light chain 3f (MLC3f) content. In the current study, we first established recombinant adenovirus (rAd) gene transfer technology to increase MLC3f content in semimembranosus muscles of aged rats. We hypothesized that 1) age-induced decline in Vo would be associated with a decrease in relative MLC3f content and 2) increasing MLC3f content via rAd would restore Vo in myosin heavy chain (MHC) type II fibers. Vo (3.2±0.2 vs. 2.2±0.2 FL/s), %MLC3f (13.6±1.0 vs. 3.7±0.3%) and MLC3f/MLC2f ratio (0.28±0.04 vs. 0.10±0.01) decreased with age in type II single fibers. Increasing %MLC3f to 8.0% and MLC3f/MLC2f ratio to 0.20 via rAd provided significant protection against age-induced reduction in Vo without causing muscle damage or influencing single fiber diameter, force and MHC composition. To the best of our knowledge, these are the first data to demonstrate affirmative effects of MLC3f against slowing of contraction in aged skeletal muscle. RO1AG017768

Rad is a p53 direct transcriptional target that inhibits cell migration and is frequently silenced in lung carcinoma cells

The p53 tumor suppressor exerts its function mainly as a transcriptional activator. Here we show that the Ras-related small GTPase Rad, an inhibitor of Rho kinase, is a direct transcriptional target of p53. Expression of Rad messenger RNA (mRNA) and protein was induced by DNA damage in a p53-dependent manner. The -2934/-2905-bp Rad promoter region, to which p53 bound, was required for p53-mediated Rad gene activation. Treatment by DNA damaging agents increased p53 occupancy and histone acetylation in the region of Rad promoter containing the p53-binding site. Expression of Rad diminished the inhibitory phosphorylation at Ser3 of cofilin, a regulator of actin dynamics, and suppressed migration and invasiveness of cancer cells. Knockdown of Rad promoted cell migration and alleviated the p53-mediated migration suppression. Frequent loss of Rad mRNA and protein expression was observed in non-small cell lung carcinoma tissues. Together our results reveal a mechanism that p53 may inhibit cell migration by disrupting actin dynamics via Rad activation and implicate a tumor suppressor role of Rad in lung cancer.

「きぼう」での宇宙放射線研究Rad Gene報告2: 低線量宇宙放射線被ばくの証拠―DSBの飛跡と放射線適応応答－

「きぼう」での宇宙放射線研究Rad Gene報告2: 低線量宇宙放射線被ばくの証拠―DSBの飛跡と放射線適応応答－

「きぼう」での宇宙放射線研究Rad Gene報告1: 宇宙環境に曝露されたヒト培養系におけるp53依存性遺伝子およびタンパク質発現

「きぼう」での宇宙放射線研究Rad Gene報告1: 宇宙環境に曝露されたヒト培養系におけるp53依存性遺伝子およびタンパク質発現

The First Life Science Experiments in ISS: Reports of "Rad Gene"-Space Radiation Effects on Human Cultured Cells-

To clarify the biological effects of space environment, especially space radiations, a proposal of Rad Gene was performed as the first life science experiment with two human lymphoblastoid cell lines bearing wild-type p53 gene (wtp53) and mutated p53 gene (mp53) in an International Space Station (ISS) for 133 days. We scheduled four projects: (1) DNA damage induced by space radiations including the high linear energy transfer (LET) particles was detected as a track of γH2AX foci in the nuclei of these frozen cells. (2) To examine the biological effects of microgravity and space radiations on gene and protein expression of p53-dependent regulated genes, these cells were grown under microgravity and 1 gravity in ISS, and on ground for 8 days and analyzed by DNA and protein arrays. (3) p53-Dependent regulated genes were analyzed in the cultured cells after spaceflight at frozen state exposed to space radiations. (4) To clarify the effects of space radiations on the radio-adaptive response, the space flown cells at frozen state were cultured, and then exposed to challenging X-ray-irradiation. All of the radio-adaptive responses of cell killing, apoptosis, chromosomal aberrations and mutations were found only in wtp53 cells, but not in the mp53 cells.

NAB2 Represses Transcription by Interacting with the CHD4 Subunit of the Nucleosome Remodeling and Deacetylase (NuRD) Complex

Early growth response (EGR) transactivators act as critical regulators of several physiological processes, including peripheral nerve myelination and progression of prostate cancer. The NAB1 and NAB2 (NGFI-A/EGR1-binding protein) transcriptional corepressors directly interact with three EGR family members (Egr1/NGFI-A/zif268, Egr2/Krox20, and Egr3) and repress activation of their target promoters. To understand the molecular mechanisms underlying NAB repression, we found that EGR activity is modulated by at least two repression domains within NAB2, one of which uniquely requires interaction with the CHD4 (chromodomain helicase DNA-binding protein 4) subunit of the NuRD (nucleosome remodeling and deacetylase) chromatin remodeling complex. Both NAB proteins can bind either CHD3 or CHD4, indicating that the interaction is conserved among these two protein families. Furthermore, we show that repression of the endogenous Rad gene by NAB2 involves interaction with CHD4 and demonstrate colocalization of NAB2 and CHD4 on the Rad promoter in myelinating Schwann cells. Finally, we show that interaction with CHD4 is regulated by alternative splicing of the NAB2 mRNA.

A Point Mutation of Adh1 Gene is Involved in the Repression of Coleoptile Elongation under Submergence in Rice

In higher plants, alcoholic fermentation is required to supply NAD+ to the glycolytic pathway, which is responsible for ATP synthesis under anaerobic conditions. Matsumura et al. (1995) previously isolated the reduced adh activity (rad) mutant in rice, in which elongation of the coleoptile is repressed under submergence. However, the rad gene had not been characterized. In the present study, we observed that, in the coleoptile, the Adh1 mRNA levels were comparable between the rad mutant and the wild type cultivar Kinmaze, while the amount of ADH1 protein was much lower in the rad mutant than in the wild type. Sequencing showed that G106 of the Adh1 gene of Kinmaze was replaced with A in the rad mutant, resulting in an E36K substitution in the deduced amino acid sequence. A genotyping experiment using F2 plants from a cross of the rad mutant with the indica cultivar Kasalath indicated that the point mutation was involved in the repression of coleoptile elongation. Furthermore, since the reduced ADH activity appeared to cause an ATP deficiency, elongation of the coleoptile was repressed in the submerged rad mutant.

Rad GTPase Attenuates Vascular Lesion Formation by Inhibition of Vascular Smooth Muscle Cell Migration

Rad (Ras associated with diabetes) GTPase is a prototypic member of a new subfamily of Ras-related GTPases with unique structural features, although its physiological role remains largely unknown. In the present study, we characterized the Rad function in vascular smooth muscle cells (VSMCs) and the influence of adenovirus-mediated Rad (Ad-Rad) gene delivery on vascular remodeling after experimental angioplasty. We documented for the first time that neointimal formation using balloon-injured rat carotid arteries was associated with a significant increase in Rad expression as determined by immunohistochemistry and quantitative real-time reverse-transcriptase polymerase chain reaction. The levels of Rad expression in VSMCs were highly induced by platelet-derived growth factor and tumor necrosis factor-alpha. Morphometric analyses 14 days after injury revealed significantly diminished neointimal formation in the Ad-Rad-treated carotid arteries compared with Ad-GFP or PBS controls, whereas the mutated form of Rad GTPase, which can bind GDP but not GTP, increased neointimal formation. Overexpression of Rad significantly inhibited the attachment and migration of VSMCs. In addition, Rad expression dramatically reduced the formation of focal contacts and stress fibers in VSMCs by blocking the Rho/ROK signaling pathway. Our data clearly identified Rad GTPase as a novel and critical mediator that inhibits vascular lesion formation. Manipulation of the Rad signaling pathway may provide new therapeutic approaches that will limit vascular pathological remodeling.

Development of a formulation that enhances gene expression and efficacy following intraperitoneal administration in rabbits and mice

We conducted a series of experiments to determine if intraperitoneal (IP) delivery of recombinant adenovirus (rAd)-based therapies is improved through carrier vehicle selection, and compared an icodextrin solution (a high molecular weight dextrin with a prolonged peritoneal cavity residence time) with a standardized phosphate buffered saline (PBS) delivery solution. In vitro, comparative adenovirus particle concentration determination (27h) and bioactivity assay (24h) indicated equivalent compatibility with icodextrin or PBS. In vivo, rabbits treated IP (100 ml) with rAd-βgal 1 × 109 P/ml in icodextrin showed improved transgene expression throughout the peritoneal wall compared to rAd-βgal in PBS. In PC-3 tumor-bearing mice treated IP with 5 × 109 P/0.5ml or 1 × 1010 P/0.5 ml rAd-βgal, transgene expression was significantly enhanced (p < 0.01) with icodextrin compared to PBS in both tumor specimens and peritoneal wall. In subsequent studies we compared prolongation of survival in intraperitoneal PC-3 and MDAH-2774 human xenograft tumor models in nude mice using rAd-p53 in icodextrin or PBS in multi-dose ranging (1 × 108 to 1 × 1010 P) experiments. The icodextrin formulation alone significantly increased rAd-p53 mediated survival (p < 0.05). In animals, these results show that IP rAd gene therapy can be improved with the use of icodextrin, and suggest that prolonged retention and distribution in the peritoneal cavity is an important factor.

A nuclear tale of two yeasts: The Yeast Nucleus

ABSTRACT Without question, numerous studies in yeast and mammals have revealed a striking commonality of underlying mechanisms that govern basic biological operations. Perhaps the most famous example from recent years has been the recognition that genes required for maintaining the yeast genome play a critical role in preventing cancer in humans. However, examining the molecular differences -the variations on a common theme, so to speak -can also be useful for understanding core biological processes. These ideas are the foundation for The Yeast Nucleus, a valuable contribution to Oxford University Press’s ‘Frontiers in Molecular Biology’ series. The textbook compares and contrasts various nuclear processes in budding yeast (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe), pointing out the similarities - and differences -that make these two somewhat unrelated yeasts the dominant model systems for studying fundamental eukaryotic processes.

Specific depletion of human anti-adenovirus antibodies facilitates transduction in an in vivo model for systemic gene therapy.

Recombinant adenoviral (rAd) vectors are capable of mediating high-efficiency gene transfer in vivo. Under conditions requiring systemic administration, however, the use of rAd vectors can be problematic due to the presence of circulating anti-adenovirus antibodies developed either through natural infection or during the course of treatment. We developed a passive immunization model in SCID/Beige mice to assess the effect of human and mouse anti-adenovirus antibodies on systemic administration of a rAd vector expressing beta-galactosidase (rAd-betagal). In this model, the in vitro neutralizing activity of human or mouse antibodies used for passive immunization correlated well with inhibition of transduction of the liver following i.v. administration of rAd-betagal. Depletion of antibodies to individual adenovirus structural proteins (hexon, penton, fiber) by affinity chromatography demonstrated that antibodies to each of the three virion components contributed to neutralization of infectivity in vitro and to inhibition of transduction in vivo. Depletion of antibodies against all three structural proteins from human or mouse immune serum prior to passive immunization restored in vivo transduction activity to levels comparable to those obtained with nonimmune serum. Our data suggest that depletion of both murine and human anti-adenoviral antibodies can restore transduction in vivo during systemic rAd gene therapy in hosts previously exposed to adenovirus.

Successful interference with cellular immune responses to immunogenic proteins encoded by recombinant viral vectors.

Vectors derived from the adeno-associated virus (AAV) have been successfully used for the long-term expression of therapeutic genes in animal models and patients. One of the major advantages of these vectors is the absence of deleterious immune responses following gene transfer. However, AAV vectors, when used in vaccination studies, can result in efficient humoral and cellular responses against the transgene product. It is therefore important to understand the factors which influence the establishment of these immune responses in order to design safe and efficient procedures for AAV-based gene therapies. We have compared T-cell activation against a strongly immunogenic protein, the influenza virus hemagglutinin (HA), which is synthesized in skeletal muscle following gene transfer with an adenovirus (Ad) or an AAV vector. In both cases, cellular immune responses resulted in the elimination of transduced muscle fibers within 4 weeks. However, the kinetics of CD4(+) T-cell activation were markedly delayed when AAV vectors were used. Upon recombinant Ad (rAd) gene transfer, T cells were activated both by direct transduction of dendritic cells and by cross-presentation of the transgene product, while upon rAAV gene transfer T cells were only activated by the latter mechanism. These results suggested that activation of the immune system by the transgene product following rAAV-mediated gene transfer might be easier to control than that following rAd-mediated gene transfer. Therefore, we tested protocols aimed at interfering with either antigen presentation by blocking the CD40/CD40L pathway or with the T-cell response by inducing transgene-specific tolerance. Long-term expression of the AAV-HA was achieved in both cases, whereas immune responses against Ad-HA could not be prevented. These data clearly underline the importance of understanding the mechanisms by which vector-encoded proteins are recognized by the immune system in order to specifically interfere with them and to achieve safe and stable gene transfer in clinical trials.

Analysis of trinucleotide-repeat combination polymorphism at the rad gene in patients with type 2 diabetes mellitus

A combined (GTT)n (ATT)n trinucleotide-repeat polymorphism designated as RAD1 has been identified at intron 2 of the rad gene on chromosome 16q. An association between the total length of the RAD1 locus and type 2 diabetes has been shown in white American subjects, but not in Finns. We genotyped 115 Japanese patients with type 2 diabetes and 114 nondiabetic control subjects at the RAD1 locus by the direct sequencing method, and found 16 RAD1 alleles composed of various combinations of GTTs and ATTs. Allele 14 consisting of four GTTs and seven ATTs accounted for the majority in both control subjects and diabetic patients, suggesting that RAD1 polymorphism is not a major genetic component for susceptibility to common forms of diabetes in the Japanese. There was no significant association between total repeat length and diabetes. However, the frequency of minor alleles containing five GTTs or three GTTs was significantly higher in diabetic patients versus nondiabetic subjects (4.8% v 0.9%, P = .012). Thus, genetic variability at the rad gene in linkage disequilibrium with RAD1 could be associated with a predisposition to type 2 diabetes in the Japanese population.

Hus1p, a conserved fission yeast checkpoint protein, interacts with Rad1p and is phosphorylated in response to DNA damage.

The hus1+ gene is one of six fission yeast genes, termed the checkpoint rad genes, which are essential for both the S-M and DNA damage checkpoints. Classical genetics suggests that these genes are required for activation of the PI-3 kinase-related (PIK-R) protein, Rad3p. Using a dominant negative allele of hus1+, we have demonstrated a genetic interaction between hus1+ and another checkpoint rad gene, rad1+. Hus1p and Rad1p form a stable complex in wild-type fission yeast, and the formation of this complex is dependent on a third checkpoint rad gene, rad9+, suggesting that these three proteins may exist in a discrete complex in the absence of checkpoint activation. Hus1p is phosphorylated in response to DNA damage, and this requires rad3+ and each of the other checkpoint rad genes. Although there is no gene related to hus1+ in the Saccharomyces cerevisiae genome, we have identified closely related mouse and human genes, suggesting that aspects of the checkpoint control mechanism are conserved between fission yeast and higher eukaryotes.

Role of Schizosaccharomyces pombe RecQ homolog, recombination, and checkpoint genes in UV damage tolerance.

The cellular responses to DNA damage are complex and include direct DNA repair pathways that remove the damage and indirect damage responses which allow cells to survive DNA damage that has not been, or cannot be, removed. We have identified the gene mutated in the rad12.502 strain as a Schizosaccharomyces pombe recQ homolog. The same gene (designated rqh1) is also mutated in the hus2.22 mutant. We show that Rqhl is involved in a DNA damage survival mechanism which prevents cell death when UV-induced DNA damage cannot be removed. This pathway also requires the correct functioning of the recombination machinery and the six checkpoint rad gene products plus the Cdsl kinase. Our data suggest that Rqh1 operates during S phase as part of a mechanism which prevents DNA damage causing cell lethality. This process may involve the bypass of DNA damage sites by the replication fork. Finally, in contrast with the reported literature, we do not find that rqh1 (rad12) mutant cells are defective in UV dimer endonuclease activity.

Molecular cloning of the human rad gene: gene structure and complete nucleotide sequence

We have isolated and sequenced human genomic DNA clones encoding the Ras-related GTP-binding protein, Rad. The gene spans 3.75 kb and consists of five exons and four introns. Translation initiates from the first of two in-frame methionine residues in the second exon. Several potential transcription cis-elements were revealed throughout the 1.7 kb 5′-flanking region, including ‘E box’ and CArG binding sites for regulators of transcription in muscle.

Polymorphism at the rad gene is not associated with NIDDM in Finns

Polymorphism at the rad gene is not associated with NIDDM in Finns