Walking Before and During a Sea Voyage

In this article More Share Options Abstract Stationary and moving surfaces impose different constraints on walking. In this study we investigated within-participants differences between walking on a ship before (at the dock) and during (at sea) a sea voyage. Four individuals participated in the study. While on the ship they wore a tri-axial accelerometer (ActiGraph GT3X+; ActiGraph LLC, Pensacola, FL) on their waists. Activity data were sampled at 30 Hz. Data were collected on the day before the voyage began and on several days at sea. The number of steps per day was greater at the dock than at sea. The net resultant force per day also was greater at the dock than at sea. However, resultant force per step was greater at sea (79.97 ± 8.50 vector magnitude counts/step) than on land (62.94 ± 10.03 vector magnitude counts/step). In addition, we observed variations in resultant force per step across days at sea. Ship motion decreased overall activity but increased the force per step

Sources and Structures of Mitotic Crossovers That Arise When BLM Helicase Is Absent in Drosophila

The Bloom syndrome helicase, BLM, has numerous functions that prevent mitotic crossovers. We used unique features of Drosophila melanogaster to investigate origins and properties of mitotic crossovers that occur when BLM is absent. Induction of lesions that block replication forks increased crossover frequencies, consistent with functions for BLM in responding to fork blockage. In contrast, treatment with hydroxyurea, which stalls forks, did not elevate crossovers, even though mutants lacking BLM are sensitive to killing by this agent. To learn about sources of spontaneous recombination, we mapped mitotic crossovers in mutants lacking BLM. In the male germline, irradiation-induced crossovers were distributed randomly across the euchromatin, but spontaneous crossovers were nonrandom. We suggest that regions of the genome with a high frequency of mitotic crossovers may be analogous to common fragile sites in the human genome. Interestingly, in the male germline there is a paucity of crossovers in the interval that spans the pericentric heterochromatin, but in the female germline this interval is more prone to crossing over. Finally, our system allowed us to recover pairs of reciprocal crossover chromosomes. Sequencing of these revealed the existence of gene conversion tracts and did not provide any evidence for mutations associated with crossovers. These findings provide important new insights into sources and structures of mitotic crossovers and functions of BLM helicase

Asynchronous replication, mono-allelic expression, and long range Cis-effects of ASAR6.

Mammalian chromosomes initiate DNA replication at multiple sites along their length during each S phase following a temporal replication program. The majority of genes on homologous chromosomes replicate synchronously. However, mono-allelically expressed genes such as imprinted genes, allelically excluded genes, and genes on female X chromosomes replicate asynchronously. We have identified a cis-acting locus on human chromosome 6 that controls this replication-timing program. This locus encodes a large intergenic non-coding RNA gene named Asynchronous replication and Autosomal RNA on chromosome 6, or ASAR6. Disruption of ASAR6 results in delayed replication, delayed mitotic chromosome condensation, and activation of the previously silent alleles of mono-allelic genes on chromosome 6. The ASAR6 gene resides within an ∼1.2 megabase domain of asynchronously replicating DNA that is coordinated with other random asynchronously replicating loci along chromosome 6. In contrast to other nearby mono-allelic genes, ASAR6 RNA is expressed from the later-replicating allele. ASAR6 RNA is synthesized by RNA Polymerase II, is not polyadenlyated, is restricted to the nucleus, and is subject to random mono-allelic expression. Disruption of ASAR6 leads to the formation of bridged chromosomes, micronuclei, and structural instability of chromosome 6. Finally, ectopic integration of cloned genomic DNA containing ASAR6 causes delayed replication of entire mouse chromosomes

Delineation of the critical region required for DRT on chromosome 6.

<p>Cells containing either an ∼47 kb deletion (ΔAAV-1d) or an ∼76 kb deletion (Δ175-23a) at the <i>ASAR6</i> locus (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003423#pgen-1003423-g007" target="_blank">Figure 7A</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003423#pgen.1003423.s005" target="_blank">S5A</a>) were incubated with BrdU for 4.5 hours, harvested, and processed for FISH using a chromosome 6 paint (red) as probe and for BrdU incorporation using an antibody against BrdU (green). The DNA was stained with DAPI (blue). A) An example of this analysis from the ΔAAV-1d (∼47 kb deletion). The two chromosome 6 s are indicated with arrows, and arbitrarily assigned i or ii. B) The two chromosome 6 s from panel A, were cut out and aligned with each color displayed separately or in combination. C) Pixel intensity profiles of the BrdU incorporation (green), and DAPI (blue) staining along the two chromosome 6 s from panel A. D) Quantification of the BrdU incorporation in chromosome 6 s. The red and blue bars represent the two chromosomes identified by the chromosome 6 paint in six different cells. E) An example of this analysis from the Δ175-23a cells (∼76 kb deletion). The two chromosome 6 s are indicated with arrows, and arbitrarily assigned i or ii. F) The two chromosome 6 s from panel E, were cut out and aligned with each color displayed separately. G) Pixel intensity profiles of the BrdU incorporation (green), and DAPI (blue) staining along the two chromosome 6 s from panel E. H) Quantification of the BrdU incorporation in chromosome 6 s. The red and blue bars represent the two chromosomes identified by the chromosome 6 paint in six different cells, the late replicating chromosome contains a deletion of ∼76 kb <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003423#pgen.1003423-Stoffregen1" target="_blank">[9]</a>.</p

Phenotypes associated with gene disruption.

<p>1. Breger et al 2005.</p><p>2. Stoffregen et al 2011.</p><p>3. Diaz-Perez et al 2006.</p

Ectopic integration of <i>ASAR6</i> results in delayed replication of mouse chromosomes.

<p>A) Schematic representation of the <i>ASAR6</i> BAC (RP11-767E7) used for integration into mouse chromosomes. The approximate locations of the loxP integration sites (red triangles) in P175 cells, deletions [in kilobases (Δkb)], and the location of the ASAR6 BAC (green) are indicated. The ASAR6 BAC was modified using recombineering to contain a deletion of the ∼29 kb (red) critical region identified by our deletion analysis. B–E) Cells containing a multicopy array of the <i>ASAR6</i> BAC integrated into mouse chromosome 3 were incubated with BrdU for 2.5 hours, harvested for mitotic cells, and processed for FISH using the <i>ASAR6</i> BAC (RP11-767E7) plus a mouse chromosome 3 BAC (RP23-430A13) as probes (both in red), and for BrdU incorporation using an antibody against BrdU (green). The DNA was stained with DAPI (blue). The chromosome 3 s are indicated by arrows. C) The chromosome 3 s from panel B, were cut out and aligned with each color displayed separately or in combination. D) Pixel intensity profiles of the BrdU incorporation (green), and DAPI (blue) staining along the chromosome 3 s from panel B are shown. Note that the chromosome 3 with the ASAR6 BAC integration (*) contains much more BrdU incorporation, indicating that it displays delayed replication. E) Quantification of the BrdU incorporation in six different cells. The red bars represent the chromosome containing the ASAR6 BAC and the blue bars represent the normal chromosome 3 s in six different cells. Most of the cells in this clone contained three chromosome 3 s. The values represent the total number of pixels (area×intensity)×1000. F–I) Cells containing a multicopy array of the <i>ASAR6</i> BAC containing an ∼29 kb deletion (see panel A, RP11-767E7-Δ29 kb) integrated into mouse chromosome 1 were incubated with BrdU for 3.0 hours, harvested for mitotic cells, and processed for FISH using the <i>ASAR6</i> BAC (RP11-767E7) plus a mouse chromosome 1 BAC (RP23-34K7) as probes (both in red), and for BrdU incorporation using an antibody against BrdU (green). The DNA was stained with DAPI (blue). The chromosome 1 s are indicated with arrows. The chromosome with the <i>ASAR6</i> BAC is marked with an asterisk (*). G) The chromosome 1 s from panel F, were cut out and aligned with each color displayed separately or in combination. H) Pixel intensity profiles of the BrdU incorporation (green), and DAPI (blue) staining along the chromosome 1 s from panel F are shown. Note that the chromosome 1 containing the ASAR6 BAC (Δ29 kb) shows similar BrdU incorporation as the normal chromosome 1. I) Quantification of the BrdU incorporation in six different cells. The red bars represent the chromosome containing the ASAR6 BAC (Δ29 kb) and the blue bars represent the normal chromosome 1 s in six different cells. The values represent the total number of pixels (area×intensity)×1000.</p

Coordinated asynchronous replication timing by single-double assay.

*<p>Indicates that the data was from Stoffregen et al. 2011.</p

Disruption of <i>ASAR6</i> leads to instability of chromosome 6.

<p>(A) An example of a mitotic spread containing three different translocations involving chromosome 6. Cells containing an engineered deletion of <i>ASAR6</i> were processed for FISH using a chromosome 6 paint as probe (green). The DNA was stained with propidium iodide. The arrows mark the locations of three different rearrangements involving chromosome 6. B) Partial karyotypes from three different mitotic spreads (1–3). The DNA was stained with propidium iodide. Notice that each cell contained different rearrangements involving chromosome 6. C) An example of a bridged chromosome between two daughter nuclei. Cells containing a disruption of ASAR6 were processed for FISH using a chromosome 6 centromeric probe (red). The DNA was stained with DAPI. Arrows mark the sites of hybridization to the chromosome 6 centromeric probe on the bridged chromosome. Chromosome bridges involving chromosome 6 were detected in approximately 1/500 metaphase spreads. D–G) Examples of micronuclei derived from chromosome 6. Cells containing a deletion of ASAR6 were processed for FISH using a chromosome 6 paint as probe (red). The DNA was stained with DAPI. Arrows mark micronuclei that hybridized to the chromosome 6 probe. Asterisks mark micronuclei that did not hybridize to the chromosome 6 paint.</p

Mono-allelic expression of <i>ASAR6</i> in human PBLs.

<p>A–I). RNA-DNA FISH for expression of <i>ASAR6</i>. PBLs were subjected to RNA FISH (green) using a Fosmid (G248P86031A6) probe for <i>ASAR6</i>. Slides were subsequently re-fixed and processed for DNA FISH (red) using BAC RP11-959I6, located distal to <i>FUT9</i> (BAC#4 in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003423#pgen-1003423-g001" target="_blank">Figure 1H</a>). In regions of the slide where the FISH worked well, the RNA FISH probe detected a positive signal in >80% of the cells. DNA was stained with DAPI. Arrows mark the location of the RNA signals. Two sites of RNA hybridization were detected in <5% of cells (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003423#pgen.1003423.s004" target="_blank">Figure S4</a> for each probe in separate images). Panel G shows an example of bi-allelic expression of ASAR6. J) <i>ASAR6</i> RNA is not polyadenlylated. Total RNA extracted from P175 cells was subjected to two rounds of Poly A selection followed by reverse transcriptase reactions (RT) in the presence (+) or absence (−) of reverse transcriptase followed by semi-quantitative PCR using primers to histone H2A, <i>MANEA</i>, or <i>ASAR6</i>. Genomic DNA was used as positive control. The primers used to detect <i>MANEA</i> cDNA span an intron and therefore do not result in a product in the genomic DNA lane. Four independent PCRs, from four different regions of ASAR6 (each spanning a different SNP; rs4645429, rs9321478, rs34875992, rs4516948) were used to detect ASAR6 RNA. The sizes of the DNA ladder (M) are indicated in base pairs. K) <i>ASAR6</i> is transcribed by RNA Polymerase II. P175 cells were exposed to 20 ug/mL of α-amanitin for 0, 5, and 10 hours. Total RNA was subjected to reverse transcriptase reactions (RT) in the presence (+) or absence (−) of reverse transcriptase followed by semi-quantitative PCR using primers to 45S RNA, a tRNA, <i>P300</i> and <i>ASAR6</i>. The sizes of the DNA ladder (M) are indicated in base pairs. Two independent PCRs, from two different regions of ASAR6 (associated with SNPs rs34875992 [top] and rs9321478 [bottom]) were used to detect ASAR6 RNA.</p