Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Effect of RNA injection on somite number in stage 24–28 embryos.

<p>Effect of RNA injection on somite number in stage 24–28 embryos.</p

A compilation of results from microarray analyses of temporal expression of Notch, WNT, and circadian genes in the somites [34] and suprachiasmatic nucleus, liver, and heart [35], [36], [37], [38].

<p>*CREB1 phosphorylation rhythm in <i>Xenopus</i> retina <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108266#pone.0108266-Li1" target="_blank">[42]</a>.</p><p>A compilation of results from microarray analyses of temporal expression of Notch, WNT, and circadian genes in the somites <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108266#pone.0108266-Curran1" target="_blank">[34]</a> and suprachiasmatic nucleus, liver, and heart <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108266#pone.0108266-Dequeant1" target="_blank">[35]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108266#pone.0108266-Panda1" target="_blank">[36]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108266#pone.0108266-Storch1" target="_blank">[37]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108266#pone.0108266-Oishi1" target="_blank">[38]</a>.</p

Reduction in xBMAL1 and xNOCTURNIN protein by morpholino injection.

<p>Both cells of a two celled embryos were injected with 1 ng Control Morpholino (Ctrl; 2 ng total), 1 ng or 500 pg of xBmal1 MO (Bmal1; 2 ng and 1 ng total) and 1 ng of <i>xNocturnin</i> MO (Noc; 2 ng total). Significant reduction of xBMAL1 protein (69Kd) was observed with injection of 2 ng or 1 ng <i>xBmal1</i> MO compared to control MO injection (0.11 and 0.26 relative to control MO injected protein levels). An approximate 50% reduction of NOCTURNIN (43Kd, indicated) protein was observed when embryos were injected with a total of 1 ng <i>xNocturnin</i> MO (0.49 relative to control MO injected protein levels). The Nocturnin antibody also recognizes a larger (62Kd) band which likely represents a postranslationally modified form of xNOCTURNIN <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108266#pone.0108266-Green2" target="_blank">[28]</a>. Alpha tubulin (100Kd) was used as a loading control for each lane.</p

Overexpression of xBMAL1 or xNOCTURNIN protein results in larger, disorganized somites with disrupted somite boundaries.

<p>Panel A represents the results of analyzing paired somite area (uninjected side vs injected) using NIH Image J. The average ratio of the area of injected to uninjected sides of the embryos analyzed is shown with error bars representing standard deviation. A significant difference in area between 150 pg <i>xBmal1</i> RNA injection (*; p<0.02) and 500 pg <i>xNocturnin</i> RNA injection was observed (*; ANOVA posthoc LSD compared to control MO, p<0.009). Panels B-G compare muscle actin (12/101) of the uninjected (B, C, F) and injected sides (D, E, G) of embryos overexpressing xBMAL1 (D, 500 pg; G, 150 pg) or xNOCTURNIN (E, 500 pg). All images were taken at the same magnification (scale bar = 10 µm).</p

<i>xClock</i>, <i>xCry1</i>, and <i>xCry2</i> are expressed in developing somites of tailbud stage embryos.

<p>Expression of each gene in the whole embryo and somites is provided. Panels A and B show <i>xClock</i> expression in the developing somites of a stage 35/36 embryo. Panels C and D show <i>xCry1</i> expression in the developing somites of a stage 37/38 embryo. Panels E and F show <i>xCry2</i> expression in a stage 37/38 embryo. White arrow heads indicate the anterior-posterior borders at the ventral extent of one somite.</p

Circadian Genes, <i>xBmal1</i> and <i>xNocturnin</i>, Modulate the Timing and Differentiation of Somites in <i>Xenopus laevis</i>

<div><p>We have been investigating whether <i>xBmal1</i> and <i>xNocturnin</i> play a role in somitogenesis, a cyclic developmental process with an ultradian period. Previous work from our lab shows that circadian genes (<i>xPeriod</i>1, <i>xPeriod2</i>, <i>xBmal1</i>, and <i>xNocturnin</i>) are expressed in developing somites. Somites eventually form the vertebrae, muscles of the back, and dermis. In <i>Xenopus</i>, a pair of somites is formed about every 50 minutes from anterior to posterior. We were intrigued by the co-localization of circadian genes in an embryonic tissue known to be regulated by an ultradian clock. Cyclic expression of genes involved in <i>Notch</i> signaling has been implicated in the somite clock. Disruption of <i>Notch</i> signaling in humans has been linked to skeletal defects in the vertebral column. We found that both depletion (morpholino) and overexpression (mRNA) of xBMAL1 protein (bHLH transcription factor) or xNOCTURNIN protein (deadenylase) on one side of the developing embryo led to a significant decrease in somite number with respect to the untreated side (p<0.001). These manipulations also significantly affect expression of a somite clock component (<i>xESR9</i>; p<0.05). We observed opposing effects on somite size. Depletion of xBMAL1 or xNOCTURNIN caused a statistically significant decrease in somite area (quantified using NIH ImageJ; p<0.002), while overexpression of these proteins caused a significant dose dependent increase in somite area (p<0.02; p<0.001, respectively). We speculate that circadian genes may play two separate roles during somitogenesis. Depletion and overexpression of xBMAL1 and NOCTURNIN both decrease somite number and influence expression of a somite clock component, suggesting that these proteins may modulate the timing of the somite clock in the undifferentiated presomitic mesoderm. The dosage dependent effects on somite area suggest that xBMAL1 and xNOCTURNIN may also act during somite differentiation to promote myogenesis.</p></div