Functional analysis of the tandem-duplicated P450 genes SPS/BUS/CYP79F1 and CYP79F2 in glucosinolate biosynthesis and plant development by Ds transposition-generated double mutants

A significant fraction (approximately 17%) of Arabidopsis genes are members of tandemly repeated families and pose a particular challenge for functional studies. We have used the Ac-Ds transposition system to generate single- and double-knockout mutants of two tandemly duplicated cytochrome P450 genes, SPS/BUS/CYP79F1 and CYP79F2. We have previously described the Arabidopsis supershoot mutants in CYP79F1 that exhibit massive overproliferation of shoots. Here we use a cytokinin-responsive reporter ARR5::uidA and an auxin-responsive reporter DR5::uidA in the sps/cyp79F1 mutant to show that increased levels of cytokinin, but not auxin, correlate well with the expression pattern of the SPS/CYP79F1 gene, supporting the involvement of this gene in cytokinin homeostasis. Further, we isolated Ds gene trap insertions in the CYP79F2 gene, and find these mutants to be defective mainly in the root system, consistent with a root-specific expression pattern. Finally, we generated double mutants in CYP79F1 and CYP79F2 using secondary transpositions, and demonstrate that the phenotypes are additive. Previous biochemical studies have suggested partially redundant functions for SPS/CYP79F1 and CYP79F2 in aliphatic glucosinolate synthesis. Our analysis shows that aliphatic glucosinolate biosynthesis is completely abolished in the double-knockout plants, providing genetic proof for the proposed biochemical functions of these genes. This study also provides further demonstration of how gluconisolate biosynthesis, regarded as secondary metabolism, is intricately linked with hormone homeostatis and hence with plant growth and development.     

Sebaceous gland adenoma in a rhesus monkey (Macaca mulatta)

A tumor mass was identified below the shoulder region of a 5-year-old male rhesus monkey (Macaca mulatta). The mass was excised and diagnosed as sebaceous gland adenoma based on the microscopic findings. Morphologically it appeared as an elevated, dome-shaped, circumscribed mass of 3.6 x 2.8 x 3.2 cm in dimension with tan speckled color. Histologically, the tumor was composed of mature, sebaceous cells (sebocytes), basal cells arranged in a mass of irregular shapes and sizes, with a characteristic appearance of poly or multilobular structure. Sebocytes were well differentiated with foamy cytoplasm in the center of the lobules and poorly or undifferentiated densely staining basal cells in the periphery of the lobules. Cellular changes in the adjacent lymph node included hyperplasia of plasma cells, macrophages and lymphoid elements with typical mitosis.     

Novel glycine transporter type-2 reuptake inhibitors. Part 2: beta- and gamma-amino acid derivatives

Several beta- and gamma-amino acid derivatives were prepared as glycine transport inhibitors and their ability to block the uptake of [(14)C]-glycine in COS7 cells transfected with human glycine transporter-2 (hGlyT-2) were evaluated. A range of lipophilic side chains were tolerated in the beta-amino acid series (i.e., Ph, CH(2)Ph, CH(CH(3))(2), and CH(2)CH(CH(3))(2)). In the gamma-amino acid series, minimal differences in potency were observed between the alpha,beta-unsaturated analogs and the corresponding saturated derivatives. In both series, a 4-biphenyl or 4-phenoxyphenyl substituent appended to the urea or cyanogunaidine moiety was necessary for in vitro activity.     

Novel fluorescence-based screen to identify small synthetic internal ribosome entry site elements

We report here a novel fluorescent protein-based screen to identify small, synthetic internal ribosome entry site (IRES) elements in vivo. A library of bicistronic plasmids encoding the enhanced blue and green fluorescent proteins (EBFP and EGFP) separated by randomized 50-nucleotide-long sequences was amplified in bacteria and delivered into mammalian cells via protoplast fusion. Cells that received functional IRES elements were isolated using the EBFP and EGFP reporters and fluorescence-activated cell sorting, and several small IRES elements were identified. Two of these elements were subsequently shown to possess IRES activity comparable to that of a variant of the encephalomyocarditis virus IRES element in a context-independent manner both in vitro and in vivo, and these elements functioned in multiple cell types. Although no sequence or structural homology was apparent between the synthetic IRES elements and known viral and cellular IRES elements, the two synthetic IRES elements specifically blocked poliovirus (PV) IRES-mediated translation in vitro. Competitive protein-binding experiments suggested that these IRES elements compete with PV IRES-mediated translation by utilizing some of the same factors as the PV IRES to direct translation. The utility of this fluorescent protein-based screen in identifying IRES elements with improved activity as well as in probing the mechanism of IRES-mediated translation is discussed.     

Exchange of the basic domain of human immunodeficiency virus type 1 Rev for a polyarginine stretch expands the RNA binding specificity, and a minimal arginine cluster is required for optimal RRE RNA binding affinity, nuclear accumulation, and trans-activation

The Rev regulatory protein of human immunodeficiency virus (HIV) facilitates the nuclear export of unspliced and partially spliced HIV RNAs. Using a Rev:MS2 phage coat protein fusion that could be targeted to bind and activate the Rev-responsive element (RRE) RNA or heterologous MS2 phage operator RNA, we analyzed the role(s) of the arginine-rich RNA binding domain in RNA binding and transactivation. The arginine-rich domain could be functionally replaced by a stretch of nine arginines. However, polyarginine substitutions expanded the RNA binding specificity of the resultant mutant Rev protein. Polyarginine insertions in place of residues 24 to 60 that excised the RNA binding and oligomerization domains of Rev preserved the activation for MS2 RNA, but not for the RRE. A nine-arginine insertion outside of the natural context of the Rev nuclear localization signal domain was incompatible with activation of either RNA target. Insertions of fewer than eight arginines impaired RRE activation. Interrupted lysine clusters and disruption of the arginine stretch with lysine or neutral residues resulted in a similar phenotype. Some of these mutants with a null phenotype for RRE activated the heterologous MS2 RNA target. Under steady-state conditions, mutants that preserved the Rev response for RRE RNA localized to the nuclei; those with poor or no Rev response accumulated mostly in the cytoplasm. Many of the cytoplasmically resident derivatives became nuclear when leptomycin B (LMB) treatment inhibited nuclear export of nuclear export signal-containing proteins. Mutants that had a null activation potential for either RNA target were particularly resistant to LMB treatment. Abbreviated nuclear residence times and differences in RRE binding affinity may have compromised their activation potential for RRE. High-affinity binding to MS2 RNA through the intact coat protein was sufficient to overcome the short nuclear residence times and to facilitate MS2 activation by some derivatives.     

Induction of heme oxygenase-1 expression in macrophages by diesel exhaust particle chemicals and quinones via the antioxidant-responsive element

Diesel exhaust particles (DEP) contain organic chemicals that contribute to the adverse health effects of inhaled particulate matter. Because DEP induce oxidative stress in the lung and in macrophages, effective antioxidant defenses are required. One type of defense is through the expression of the antioxidant enzyme, heme oxygenase I (HO-1). HO-1 as well as phase II detoxifying enzymes are induced via antioxidant response elements (ARE) in their promoters of that gene. We show that a crude DEP total extract, aromatic and polar DEP fractions, a benzo(a)pyrene quinone, and a phenolic antioxidant induce HO-1 expression in RAW264.7 cells in an ARE-dependent manner. N-acetyl cysteine and the flavonoid, luteolin, inhibited HO-1 protein expression. We also demonstrate that the same stimuli induce HO-1 mRNA expression in parallel with the activation of the SX2 enhancer of that gene. Mutation of the ARE core, but not the overlapping AP-1 binding sequence, disrupted SX2 activation. Finally, we show that biological agents, such as oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine, could also induce HO-1 expression via an ARE-dependent mechanism. Prior induction of HO-1 expression, using cobalt-protoporphyrin, protected RAW264.7 cells against DEP-induced toxicity. Taken together, these data show that HO-1 plays an important role in cytoprotection against redox-active DEP chemicals, including quinones.     

Bioactive Peptides from Marine Ascidians and Future Drug Development–A Review

Marine ascidians are considered as one of the richest sources of bioactive compounds. The extraction and utilization of marine peptides have been attracted much attention owing to their potential health benefits. Most of the bioactive compounds from marine ascidians are already in different phases of the clinical and preclinical pipeline. They can be used in different functional and nutraceutical values due to their antineoplastic, antihypertensive, antioxidant, and antimicrobial properties. The screening in vivo and in vitro bioassays are coupled to the purification process for the exploration of its biological interest which is of great value. The growing significance to study marine natural products results from the discovery of novel pharmacological tools including potent anticancer drugs and other drugs are in clinical/pre-clinical trials. The present review highlights the recent research progress in marine ascidians’ peptides and its prospects for the future pharmaceutical development.     

Green approach for synthesis of zinc oxide nanoparticles from Andrographis paniculata leaf extract and evaluation of their antioxidant,anti-diabetic,and anti-inflammatory activities

Bioprocess and Biosystems Engineering - Bio-mediated synthesis of zinc oxide nanoparticles (ZnO NPs) was carried out by utilizing the reducing and capping potential of Andrographis paniculata leaf...     

Production of a High-Efficiency TILLING Population through Polyploidization

Targeting Induced Local Lesions in Genomes (TILLING) provides a nontransgenic method for reverse genetics that is widely applicable, even in species where other functional resources are missing or expensive to build. The efficiency of TILLING, however, is greatly facilitated by high mutation density. Species vary in the number of mutations induced by comparable mutagenic treatments, suggesting that genetic background may affect the response. Allopolyploid species have often yielded higher mutation density than diploids. To examine the effect of ploidy, we autotetraploidized the Arabidopsis (Arabidopsis thaliana) ecotype Columbia, whose diploid has been used for TILLING extensively, and mutagenized it with 50 mm ethylmethane sulfonate. While the same treatment sterilized diploid Columbia, the tetraploid M1 plants produced good seed. To determine the mutation density, we searched 528 individuals for induced mutations in 15 genes for which few or no knockout alleles were previously available. We constructed tridimensional pools from the genomic DNA of M2 plants, amplified target DNA, and subjected them to Illumina sequencing. The results were analyzed with an improved version of the mutation detection software CAMBa that accepts any pooling scheme. This small population provided a rich resource with approximately 25 mutations per queried 1.5-kb fragment, including on average four severe missense and 1.3 truncation mutations. The overall mutation density of 19.4 mutations Mb^–1 is 4 times that achieved in the corresponding diploid accession, indicating that genomic redundancy engenders tolerance to high mutation density. Polyploidization of diploids will allow the production of small populations, such as less than 2,000, that provide allelic series from knockout to mild loss of function for virtually all genes.The growing availability of whole-genome sequences is spurring functional gene studies in species where specific tools for reverse genetics are not available. However, developing suitable functional genetic resources is often challenging and expensive. For example, targeted gene inactivation through transfer DNA (T-DNA; Alonso et al., 2003) or transposable elements (Krishnan et al., 2009) typically requires 300,000 to 500,000 tagged individuals to approach saturation, even for a small genome like that of Arabidopsis (Arabidopsis thaliana), and both tools may be needed to avoid intrinsic insertional bias (Pan et al., 2005). Two other tools, RNA interference and targeted endonucleases, require transformation and cannot be scaled easily. Furthermore, transgenic plants require regulatory clearance, hindering phenotypic characterization. Targeting Induced Local Lesions in Genomes (TILLING) is a functional genomics method that discovers chemically induced mutations in populations and presents considerable advantages: it is applicable to many sexual species, it requires relatively small populations, it is not transgenic, and it can target potentially any gene (Comai and Henikoff, 2006; Wang et al., 2012). TILLING consists of mutagenesis, DNA isolation and pooling, and high-throughput mutation discovery in targeted genes. First described in Arabidopsis (McCallum et al., 2000) and Drosophila spp. (Bentley et al., 2000), it has been successfully extended to multiple model and economic species, thus becoming an important tool for functional genomics. Originally, TILLING discovered mutations through the detection of mismatched sites in PCR products (Oleykowski et al., 1998; Till et al., 2004a; Dong et al., 2009). The advent of low-cost high-throughput sequencing has added another powerful method (Rigola et al., 2009; Tsai et al., 2011). Tsai et al. (2011) recently described the use of Illumina sequencing and single-nucleotide polymorphism analysis in multidimensional pools as a method for efficient mutation discovery.TILLING efficiency (i.e. the cost of obtaining informative mutations per gene) depends on the characteristics of the population used and particularly on the mutation density (Comai and Henikoff, 2006). This, in turn, depends at least in part on the intensity used to mutagenize the target species and on its response. Mutagenesis is usually applied by the treatment of seed with a chemical mutagen in a manner that produces tolerable lethality and sterility of the treated individuals, the M1 plants, while allowing sufficient production of fertile M2 plants. M2 DNA and M3 seed are typically inventoried to preserve the resource. Optimal conditions for mutagenesis vary from species to species, and mutation densities determined by TILLING differ as much as 100-fold. Since similar treatments yield different mutation densities in different species, cellular or developmental characteristics depending on the genetic background must play a role in the outcome. The highest mutation densities were obtained in polyploids (Fig. 1), hexaploid wheat (Triticum aestivum; Slade et al., 2005; Uauy et al., 2009), and tetraploid canola (Brassica napus; Wang et al., 2008; Harloff et al., 2012), displaying the highest number at approximately 40 mutations Mb^–1 of diploid DNA (i.e. for each of the ancestral genomes of the polyploid). Allotetraploid peanut (Arachis hypogaea), on the other hand, is an exception, with about 1 mutation Mb^–1 (Knoll et al., 2011). Paleopolyploids have yielded diverse mutation rates: Brassica rapa yielded a near-polyploid-like density at 16.6 mutations Mb^–1 (Stephenson et al., 2010), while soybean (Glycine max) and maize (Zea mays) displayed lower mutation rates (Till et al., 2004b; Cooper et al., 2008) within the range of diploid responses. Arabidopsis accession Landsberg erecta (Ler) yielded about 11 mutations Mb^–1 (Martín et al., 2009), one of the highest densities described for a diploid. Comparable mutagenic treatments in Arabidopsis accession Columbia (Col-0), however, yielded about 4 mutations Mb^–1 (Greene et al., 2003).Open in a separate windowFigure 1.Effect of mutation rate and ploidy on functional discovery through TILLING. A, The probability of getting at least one severe missense mutation (assumed to be 15% of all mutations) and one knockout (KO; 5% of all mutations) is plotted versus the total mutation number identified in the coding region of a gene (redrawn from Henikoff et al. [2004]). B, The relationship between mutant yield and mutation rate is illustrated by the number of individuals in a population required to yield a given number of mutations (in A) in a 1-kb fragment. For example, considering mutations in a 1-kb coding region, the hatched blue stripe highlights how a population with a mutation rate of 2 mutations Mb^–1 requires screening more than 15,000 individuals for a 0.8 confidence of obtaining at least a single knockout. The same result can be obtained with 768 individuals of a population that has a mutation rate of 40 mutations Mb^–1, such as hexaploid wheat. The number of mutations expected for a given population and mutation density should be scaled according to the gene size. For example, for a 2-kb coding region and a mutation density of 2 mutations Mb^–1, 60 mutations are expected. A population of 15,000 individuals would yield an approximately 95% chance of at least one knockout. C, Published mutation rates in TILLING populations of different species organized according to ploidy. The vertical bar connects instances of the same species. Ai, Peanut (Knoll et al., 2011); AtC, Arabidopsis Col-0 (Greene et al., 2003); AtL, Arabidopsis Ler (Martín et al., 2009), Bn, canola (Harloff et al., 2012); Br, B. rapa (Stephenson et al., 2010); Gm, soybean (Cooper et al., 2008); Hv, barley (Hordeum vulgare; Talamè et al., 2008); Mt, Medicago truncatula (Le Signor et al., 2009); Os, rice (Oryza sativa; Till et al., 2007); Ps, pea (Pisum sativum; Dalmais et al., 2008); Ta, wheat (6x; Slade et al., 2005; Uauy et al., 2009); Td, durum wheat (Triticum durum [4x]; Slade et al., 2005; Uauy et al., 2009); Zm, maize (Till et al., 2004b). [See online article for color version of this figure.]There is no information on the molecular mechanisms underlying variable mutation yields in plants. Such knowledge should be useful for improving TILLING populations. Available data suggest a possible role for polyploidy, but not genome size, in conferring tolerance to high mutation density, a finding consistent with an early analysis (Stadler, 1929). There are at least two possible explanations for such an effect. First, genetic redundancy may shield polyploids from the deleterious consequences of mutation, making them more tolerant to mutagenic agents. Second, polyploids may be physiologically more tolerant of genotoxic treatments or more susceptible to mutagenesis. Such changes, for example, could result from adaptive changes affecting DNA repair and genome maintenance taking place after polyploidization.Here, we show that an autotetraploid derivative of Arabidopsis accession Col-0 resists an ethylmethane sulfonate (EMS) concentration that sterilizes its diploid progenitor while at the same time accumulating mutations at a density four times that previously achieved with this ecotype. We discuss the implications of this finding in the context of whole-genome duplication and functional genomics.