Sorghum Genome Sequencing by Methylation Filtration

Sorghum bicolor is a close relative of maize and is a staple crop in Africa and much of the developing world because of its superior tolerance of arid growth conditions. We have generated sequence from the hypomethylated portion of the sorghum genome by applying methylation filtration (MF) technology. The evidence suggests that 96% of the genes have been sequence tagged, with an average coverage of 65% across their length. Remarkably, this level of gene discovery was accomplished after generating a raw coverage of less than 300 megabases of the 735-megabase genome. MF preferentially captures exons and introns, promoters, microRNAs, and simple sequence repeats, and minimizes interspersed repeats, thus providing a robust view of the functional parts of the genome. The sorghum MF sequence set is beneficial to research on sorghum and is also a powerful resource for comparative genomics among the grasses and across the entire plant kingdom. Thousands of hypothetical gene predictions in rice and Arabidopsis are supported by the sorghum dataset, and genomic similarities highlight evolutionarily conserved regions that will lead to a better understanding of rice and Arabidopsis

Disruption of Diacylglycerol Kinase Delta (DGKD) Associated with Seizures in Humans and Mice

We report a female patient with a de novo balanced translocation, 46,X,t(X;2)(p11.2;q37)dn, who exhibits seizures, capillary abnormality, developmental delay, infantile hypotonia, and obesity. The 2q37 breakpoint observed in association with the seizure phenotype is of particular interest, because it lies near loci implicated in epilepsy in humans and mice. Fluorescence in situ hybridization mapping of the translocation breakpoints showed that no known genes are disrupted at Xp11.2, whereas diacylglycerol kinase delta (DGKD) is disrupted at 2q37. Expression studies in Drosophila and mouse suggest that DGKD is involved in central nervous system development and function. Electroencephalographic assessment of Dgkd mutant mice revealed abnormal epileptic discharges and electrographic seizures in three of six homozygotes. These findings implicate DGKD disruption by the t(X;2)(p11.2;q37)dn in the observed phenotype and support a more general role for DGKD in the etiology of seizures

The seventh international RASopathies symposium: Pathways to a cure-expanding knowledge, enhancing research, and therapeutic discovery.

RASopathies are a group of genetic disorders that are caused by genes that affect the canonical Ras/mitogen-activated protein kinase (MAPK) signaling pathway. Despite tremendous progress in understanding the molecular consequences of these genetic anomalies, little movement has been made in translating these findings to the clinic. This year, the seventh International RASopathies Symposium focused on expanding the research knowledge that we have gained over the years to enhance new discoveries in the field, ones that we hope can lead to effective therapeutic treatments. Indeed, for the first time, research efforts are finally being translated to the clinic, with compassionate use of Ras/MAPK pathway inhibitors for the treatment of RASopathies. This biannual meeting, organized by the RASopathies Network, brought together basic scientists, clinicians, clinician scientists, patients, advocates, and their families, as well as representatives from pharmaceutical companies and the National Institutes of Health. A history of RASopathy gene discovery, identification of new disease genes, and the latest research, both at the bench and in the clinic, were discussed

The seventh international RASopathies symposium:Pathways to a cure-expanding knowledge, enhancing research, and therapeutic discovery

RASopathies are a group of genetic disorders that are caused by genes that affect the canonical Ras/mitogen-activated protein kinase (MAPK) signaling pathway. Despite tremendous progress in understanding the molecular consequences of these genetic anomalies, little movement has been made in translating these findings to the clinic. This year, the seventh International RASopathies Symposium focused on expanding the research knowledge that we have gained over the years to enhance new discoveries in the field, ones that we hope can lead to effective therapeutic treatments. Indeed, for the first time, research efforts are finally being translated to the clinic, with compassionate use of Ras/MAPK pathway inhibitors for the treatment of RASopathies. This biannual meeting, organized by the RASopathies Network, brought together basic scientists, clinicians, clinician scientists, patients, advocates, and their families, as well as representatives from pharmaceutical companies and the National Institutes of Health. A history of RASopathy gene discovery, identification of new disease genes, and the latest research, both at the bench and in the clinic, were discussed

Secondary Structure of Predicted MiRNAs

<p>Predicted hairpin secondary structure of miRNA MIR156a from rice and the newly discovered ortholog from sorghum. The 21-nucleotide MIR156a sequence is highlighted in red.</p

Annotation of <i>Arabidopsis</i> by Sorghum MF Versus Rice Gene Sequences

<p>Shown are the number of <i>Arabidopsis</i> proteins that are matched in a TBLASTN comparison to the sorghum MF set (blue) versus the rice gene sequences (yellow). The <i>Arabidopsis</i> proteins, after having known repetitive elements removed (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030013#s3" target="_blank">Materials and Methods</a>), have been categorized as either hypothetical or known based on the definition line. <i>Arabidopsis</i> proteins were considered supported if they matched with an E-value less than or equal to 1 × 10⁻⁸. Sb, S. bicolor MF set; Osj:seq, <i>Oryza sativa japonica</i> gene sequences.</p

Genome Reduction

<p>MF reduces the sorghum genome by 66% in sampling a hypomethylated space of approximately 247 Mb (green) and filtering out 488 Mb (red) of the 735-Mb sorghum genome.</p

Methylation Status of <i>tb2</i> and Kafirin Cluster

<div><p>(A and B) Restriction maps of the <i>tb2</i> gene (A) and the kafirin consensus sequences (B) are shown. The relevant restriction sites are indicated vertically and the numbers indicate the distances scale in basepairs. Each CDS is depicted as a blue-shaded arrow, and the region assayed is indicated by a black bar. The circles depict sites that are not present in every kafirin gene, and the color represents the number of genes that do not share the site. The orange circle (5′-most HhaI site) is a site conserved in nine of 11 kafirin genes, and the red circle (3′-most PstI site) is a site present in ten of the 11.</p> <p>(C) Results from a representative methylation analysis of <i>tb2;</i> the inset depicts the template dilution standard curve used to set the threshold for the experiment. Each experiment was performed three times with four on-board replicates per assay point. The results for each of the four differentially treated reactions are depicted with different colors. Red, mock-treated; blue, mcrBC-digested; orange, HhaI-digested; and green, HhaI + mcrBC double-digest. The inset shows the standard dilution control with two replicates at each dilution. The control was used to set the threshold for detection. The specificity of each reaction was confirmed using melt-curve analysis.</p> <p>(D) Results from a representative methylation analysis of the 11 kafirin genes. The results for each of the six differentially treated reactions are the same as in (C), with the following additional digests: pink, PstI-digested; light blue, PstI + mcrBC double-digest. Notice that the mcrBC with and without PstI yields the same Ct, while HhaI + mcrBC (green) yields a higher Ct on average; suggesting additional cleavage.</p></div

CG and CNG Suppression in MF versus UF Sequences

<p>Sequences were analyzed for their mcrBC half-sites, those that overlap CG dinucleotides, and those that overlap CNG trinucleotides. The ratio of observed to expected sites is graphed for filtered (hatched) and unfiltered (white) for retrotransposons (A) and CDSs (B).</p