Cloning and analysis of the autonomous element, MuDR:Cy, of the Mutator family of maize

An autonomous Cy transposable element of Zea mays inserted at the a1 locus (a1-m5216: MuDR) was isolated genetically and molecularly cloned. Restriction mapping and sequencing results indicate that Cy (designated MuDR:Cy1) is another isolate of the autonomous element of the Mutator family, MuDR. Six defective derivative alleles of a1-m5216:MuDR were isolated genetically and molecularly cloned. Five of these a1-r alleles (a1-r5835, a1-r5431, a1-r182, a1-r5938 and a1-r5306) behave like non-autonomous elements in that their transposition can only be activated by active MuDR elements in trans. Three of these a1-r alleles have deletions that disrupt transcript mudrA. One allele has a deletion that disrupts transcript mudrB. The lesion associated with the remaining a1-r allele could disrupt either mudrA or mudrB, or both. Analysis of the a1-r alleles results suggests that both transcripts encoded by active MuDR elements, mudrA and mudrB, are necessary for autonomous transposition. The sixth allele, a1-nr5940, which does not respond to the activation of active MuDR elements, has a deletion involving transcript mudrA, the 5\u27 terminal inverted repeat of MuDR and a1 coding sequence. The origin of the a1-r and a1-nr alleles can be explained by the gap-repair model. Another MuDR:Cy element (designated MuDR:Cy2 and which maps 10 cM distal of the pr1 locus on chromosome 5) was shown to transpose at a rate of 3.3% of gametes per generation and increase its copy numbers at rates from 0 to 28.6% of MuDR-containing gametes. Genetic and molecular tests in inactive Mutator lines demonstrated that an active MuDR:Cy2 element can restore the Mu1 hypomethylation and reactivate the high forward mutation rate associated with active Mutator lines. The subsequent loss of MuDR:Cy2 via meitotic segregation resulted in increased levels of Mu1 methylation. Analysis of a novel change of state of a Mu element insertion mutation at the bz1 locus (bz1-rcy4333y) revealed that developmental regulation of MuDR activity is not responsible for the unique insertion and excision patterns associated with Mu elements. Instead, a model involving developmentally regulated gap-repair is proposed

The B73 Maize Genome: Complexity, Diversity, and Dynamics

We report an improved draft nucleotide sequence of the 2.3-gigabase genome of maize, an important crop plant and model for biological research. Over 32,000 genes were predicted, of which 99.8% were placed on reference chromosomes. Nearly 85% of the genome is composed of hundreds of families of transposable elements, dispersed nonuniformly across the genome. These were responsible for the capture and amplification of numerous gene fragments and affect the composition, sizes, and positions of centromeres. We also report on the correlation of methylation-poor regions with Mu transposon insertions and recombination, and copy number variants with insertions and/or deletions, as well as how uneven gene losses between duplicated regions were involved in returning an ancient allotetraploid to a genetically diploid state. These analyses inform and set the stage for further investigations to improve our understanding of the domestication and agricultural improvements of maize

Refinement of Light-Responsive Transcript Lists Using Rice Oligonucleotide Arrays: Evaluation of Gene-Redundancy

Studies of gene function are often hampered by gene-redundancy, especially in organisms with large genomes such as rice (Oryza sativa). We present an approach for using transcriptomics data to focus functional studies and address redundancy. To this end, we have constructed and validated an inexpensive and publicly available rice oligonucleotide near-whole genome array, called the rice NSF45K array. We generated expression profiles for light- vs. dark-grown rice leaf tissue and validated the biological significance of the data by analyzing sources of variation and confirming expression trends with reverse transcription polymerase chain reaction. We examined trends in the data by evaluating enrichment of gene ontology terms at multiple false discovery rate thresholds. To compare data generated with the NSF45K array with published results, we developed publicly available, web-based tools (www.ricearray.org). The Oligo and EST Anatomy Viewer enables visualization of EST-based expression profiling data for all genes on the array. The Rice Multi-platform Microarray Search Tool facilitates comparison of gene expression profiles across multiple rice microarray platforms. Finally, we incorporated gene expression and biochemical pathway data to reduce the number of candidate gene products putatively participating in the eight steps of the photorespiration pathway from 52 to 10, based on expression levels of putatively functionally redundant genes. We confirmed the efficacy of this method to cope with redundancy by correctly predicting participation in photorespiration of a gene with five paralogs. Applying these methods will accelerate rice functional genomics

Molecular control of HIV-1 postintegration latency: implications for the development of new therapeutic strategies

The persistence of HIV-1 latent reservoirs represents a major barrier to virus eradication in infected patients under HAART since interruption of the treatment inevitably leads to a rebound of plasma viremia. Latency establishes early after infection notably (but not only) in resting memory CD4+ T cells and involves numerous host and viral trans-acting proteins, as well as processes such as transcriptional interference, RNA silencing, epigenetic modifications and chromatin organization. In order to eliminate latent reservoirs, new strategies are envisaged and consist of reactivating HIV-1 transcription in latently-infected cells, while maintaining HAART in order to prevent de novo infection. The difficulty lies in the fact that a single residual latently-infected cell can in theory rekindle the infection. Here, we review our current understanding of the molecular mechanisms involved in the establishment and maintenance of HIV-1 latency and in the transcriptional reactivation from latency. We highlight the potential of new therapeutic strategies based on this understanding of latency. Combinations of various compounds used simultaneously allow for the targeting of transcriptional repression at multiple levels and can facilitate the escape from latency and the clearance of viral reservoirs. We describe the current advantages and limitations of immune T-cell activators, inducers of the NF-κB signaling pathway, and inhibitors of deacetylases and histone- and DNA- methyltransferases, used alone or in combinations. While a solution will not be achieved by tomorrow, the battle against HIV-1 latent reservoirs is well- underway

Cloning and analysis of the autonomous element, MuDR:Cy, of the Mutator family of maize

An autonomous Cy transposable element of Zea mays inserted at the a1 locus (a1-m5216: MuDR) was isolated genetically and molecularly cloned. Restriction mapping and sequencing results indicate that Cy (designated MuDR:Cy1) is another isolate of the autonomous element of the Mutator family, MuDR. Six defective derivative alleles of a1-m5216:MuDR were isolated genetically and molecularly cloned. Five of these a1-r alleles (a1-r5835, a1-r5431, a1-r182, a1-r5938 and a1-r5306) behave like non-autonomous elements in that their transposition can only be activated by active MuDR elements in trans. Three of these a1-r alleles have deletions that disrupt transcript mudrA. One allele has a deletion that disrupts transcript mudrB. The lesion associated with the remaining a1-r allele could disrupt either mudrA or mudrB, or both. Analysis of the a1-r alleles results suggests that both transcripts encoded by active MuDR elements, mudrA and mudrB, are necessary for autonomous transposition. The sixth allele, a1-nr5940, which does not respond to the activation of active MuDR elements, has a deletion involving transcript mudrA, the 5' terminal inverted repeat of MuDR and a1 coding sequence. The origin of the a1-r and a1-nr alleles can be explained by the gap-repair model. Another MuDR:Cy element (designated MuDR:Cy2 and which maps 10 cM distal of the pr1 locus on chromosome 5) was shown to transpose at a rate of 3.3% of gametes per generation and increase its copy numbers at rates from 0 to 28.6% of MuDR-containing gametes. Genetic and molecular tests in inactive Mutator lines demonstrated that an active MuDR:Cy2 element can restore the Mu1 hypomethylation and reactivate the high forward mutation rate associated with active Mutator lines. The subsequent loss of MuDR:Cy2 via meitotic segregation resulted in increased levels of Mu1 methylation. Analysis of a novel change of state of a Mu element insertion mutation at the bz1 locus (bz1-rcy4333y) revealed that developmental regulation of MuDR activity is not responsible for the unique insertion and excision patterns associated with Mu elements. Instead, a model involving developmentally regulated gap-repair is proposed.</p

Types and Frequencies of Sequencing Errors in Methyl-Filtered and High C(0)t Maize Genome Survey Sequences

The Maize Genome Sequencing Consortium has deposited into GenBank more than 850,000 maize (Zea mays) genome survey sequences (GSSs) generated via two gene enrichment strategies, methylation filtration and high-C(0)t (HC) fractionation. These GSSs are a valuable resource for generating genome assemblies and the discovery of single nucleotide polymorphisms and nearly identical paralogs. Based on the rate of mismatches between 183 GSSs (105 methylation filtration + 78 HC) and 10 control genes, the rate of sequencing errors in these GSSs is 2.3 × 10(−3). As expected many of these errors were derived from insufficient vector trimming and base-calling errors. Surprisingly, however, some errors were due to cloning artifacts. These G•C to A•T transitions are restricted to HC clones; over 40% of HC clones contain at least one such artifact. Because it is not possible to distinguish the cloning artifacts from biologically relevant polymorphisms, HC sequences should be used with caution for the discovery of single nucleotide polymorphisms or paramorphisms. The average rate of sequencing errors was reduced 6-fold (to 3.6 × 10(−4)) by applying more stringent trimming parameters. This trimming resulted in the loss of only 11% of the bases (15,469/144,968). Due to redundancy among GSSs this more stringent trimming reduced coverage of promoters, exons, and introns by only 0%, 1%, and 4%, respectively. Hence, at the cost of a very modest loss of gene coverage, the quality of these maize GSSs can approach Bermuda standards, even prior to assembly

Picky: oligo microarray design for large genomes

*To whom correspondence should be addressed. Motivation: Many large genomes are getting sequenced nowadays. Biologists are eager to start microarray analysis taking advantage of all known genes of a species, but existing microarray design tools were very inefficient for large genomes. Also, many existing tools operate in a batch mode that does not assure best designs. Results: PICKY is an efficient oligo microarray design tool for large genomes. PICKY integrates novel computer science techniques and the best known nearest-neighbor parameters to quickly identify sequence similarities and estimate their hybridization properties. Oligos designed by PICKY are computationally optimized to guarantee the best specificity, sensitivity and uniformity under the given design constrains. PICKY can be used to design arrays for whole genomes, or for only a subset of genes. The latter can still be screened against a whole genome to attain the same quality as a whole genome array, thereby permitting low budget, pathway-specific experiments to be conducted with large genomes. PICKY is the fastest oligo array design tool currently available to the public, requiring only a few hours to process large gene sets from rice, maize or human. Availability: PICKY is independent of any external software to execute, is designed for nonprogrammers to easily operate through a graphical user interface, and is made available for all major computing platforms (e.g., Mac, Windows and Linux) a

Monascus Adlay and Monacolin K Attenuates Arterial Thrombosis in Rats through the Inhibition of ICAM-1 and Oxidative Stress

Background/Aims: Monascus Adlay (MA) prepared from fungal fermentation of Monascus purpureus inoculating with cooked adlay contains high content of monakolin K (MK) and phenolic compounds. We explored whether MA and MK improve FeCl3-induced arterial thrombosis in rats. Methods: The rats were divided into control, FeCl3-treated rat carotid artery occlusion (TTO), TTO determined with one-week MA, and TTO determined with one-week MK. We compared MA or MK effects on oxidative stress by chemiluminescence amplification and immunohistochemistry, TTO by a transonic system, NF&#x03BA;B, ICAM-1, endoplasmic reticulum stress CHOP and Nrf2 signaling by western blotting. Results: MA or MK efficiently depressed O2-, H2O2 and HOCl levels, platelet activation and aggregation and H2O2-enhanced ICAM-1 and VCAM-1 expression in the endothelial cells. FeCl3 significantly increased NF&#x03BA;B p65, 3-nitrotyrosine, CHOP and ICAM-1 expression, and decreased nuclear Nrf2 translocation and induces arterial thrombus formation. MA or MK pretreatment significantly elongated the level of FeCl3-induced TTO compared to TTO group, significantly decreased proinflammatory NF-&#x03BA;B/ICAM-1 signaling, endoplasmic reticulum stress CHOP expression and decreased thrombotic area. MA or MK significantly preserved nuclear Nrf2 translocation. MA and MK exerted a similar protective effect in attenuating thrombus formation. Conclusions: We suggest MA is better than MK to improve FeCl3-induced arterial thrombosis