Prokaryotic Expression and Purification of Soluble Maize Ac Transposase

Transposons are mobile genetic elements that are found in all eukaryotic and prokaryotic species studied to date. The Maize Activator (Ac) transposase recognizes and excises Ac and Dissociation (Ds) elements and mediates insertion elsewhere in the genome. Insertions of Ds can cause disruption in gene sequences and hence are important functional genomics tool for tagging and cloning of unknown gene sequences. The involvement of Ac transposase (AcTPase) in Ds movement is well documented; however, protein structure and function of AcTPase is poorly understood. To express the maize AcTPase in E. coli, Ac cDNA was synthesized with an N-terminal 6xHis tag and cloned in pTrcAc expression vector. The expression cassette was induced in Rosetta2 (DE3) E. coli lines. End-point RT-PCR confirmed the integrity of AcTPase mRNA during cell culture. Autoinducing cultures grown at 37 °C produced prominent partial AcTPase products of ~40 kDa and ~70 kDa. Trypsin digestion and mass spectrometry analyses confirmed AcTPase in both the eluted peptides. When the cultures were grown at 22–25 °C for 24 h the expected ~90 kDa AcTPase soluble product was detected. The successful expression of full length AcTPase in soluble form allows further investigation of its structure and function.     

New insights into <Emphasis Type="Italic">Oryza</Emphasis> genome evolution: high gene colinearity and differential retrotransposon amplification

An ∼247-kb genomic region from FF genome of wild rice Oryza brachyantha, possessing the smallest Oryza genome, was compared to the orthologous ∼450-kb region from AA genome, O. sativa L. ssp. japonica. 37 of 38 genes in the orthologous regions are shared between japonica and O. brachyantha. Analyses of nucleotide substitution in coding regions suggest the two genomes diverged ∼10 million years ago. Comparisons of transposable elements (TEs) reveal that the density of DNA TEs in O. brachyantha is comparable to O. sativa; however, the density of RNA TEs is dramatically lower. The genomic fraction of RNA TEs in japonica is two times greater than in O. brachyantha. Differences, particularly in RNA TEs, in this region and in BAC end sequences from five wild and two cultivated Oryza species explain major genome size differences between sativa and brachyantha. Gene expression analyses of three ObDREB1 genes in the sequenced region indicate orthologous genes retain similar expression patterns following cold stress. Our results demonstrate that size and number of RNA TEs play a major role in genomic differentiation and evolution in Oryza. Additionally, distantly related O. brachyantha shares colinearity with O. sativa, offering opportunities to use comparative genomics to explore the genetic diversity of wild species to improve cultivated rice. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Data deposition: Sequence data from this article were deposited with GenBank Library under accession number DQ810282. Shibo Zhang and Yong Qiang Gu contributed equally to the work     

Thematic review series: adipocyte biology. Adipose tissue function and plasticity orchestrate nutritional adaptation

This review focuses on adipose tissue biology and introduces the concept of adipose tissue plasticity and expandability as key determinants of obesity-associated metabolic dysregulation. This concept is fundamental to our understanding of adipose tissue as a dynamic organ at the center of nutritional adaptation. Here, we summarize the current knowledge of the mechanisms by which adipose tissue can affect peripheral energy homeostasis, particularly in the context of overnutrition. Two mechanisms emerge that provide a molecular understanding for obesity-associated insulin resistance. These are a) the dysregulation of adipose tissue expandability and b) the abnormal production of adipokines. This knowledge has the potential to pave the way for novel therapeutic concepts and strategies for managing and/or correcting complications associated with obesity and the metabolic syndrome.     

Synergistic versus antagonistic actions of glutamate and glutathione: the role of excitotoxicity and oxidative stress in neuronal disease.

The etiology of various age-related neurological diseases remains unknown. Sporadic forms ofAlzheimer's, Parkinson's and Lou Gehrig's disease have been linked to environmental factors that cause neuronal cell death either by excitotoxicity or by inducing oxidative stress. Our recent studies have demonstrated that various compounds not previously associated with these diseases, i.e. methionine sulfoximine (MSO), originally isolated from 'agenized' flour, and sitosterol glucoside (BSSG), isolated from the seed of the cycad, appear to be neurotoxins, likely acting by excitotoxic mechanisms. For these compounds, the primary excitotoxic effect appears to involve glutamate release followed by NMDA receptor activation. Lactate dehydrogenase assays demonstrate that both compounds cause rapid cell death in vitro. In addition, both compounds appear to alter antioxidant defense mechanisms, acting particularly on levels of reduced glutathione (GSH). In vivo application of MSO has historically been linked to behavioral abnormalities, including seizures, in various species. Our recent experiments have demonstrated that mice fed cycad flour containing sitosterol glucoside have severe behavioral abnormalities of motor and cognitive function, as well as significant levels of neurodegeneration in cortex, hippocampus, spinal cord and other CNS regions measured post mortem. The combined weight of excitotoxic action, in concert to a decline in antioxidant defenses, induced by molecules such as methionine sulfoximine and sitosterol glucoside is hypothesized to be causal to neuronal degeneration in various neurological diseases. Understanding the mechanisms of action of these and functionally related molecules may serve to focus attention on potential neurotoxins present in the human environment. Only once such molecules have been identified, can we begin to design appropriate pharmaceutical strategies to prevent or halt the progression of the age-related neurological diseases.