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Genes required for survival in microgravity revealed by genome-wide yeast deletion collections cultured during spaceflight.

Nislow C, Lee AY, Allen PL, Giaever G, Smith A, Gebbia M, Stodieck LS, Hammond JS, Birdsall HH, Hammond TG - Biomed Res Int (2015)

Bottom Line: Spaceflight is a unique environment with profound effects on biological systems including tissue redistribution and musculoskeletal stresses.The genome-wide sensitivity profiles obtained from these treatments were then queried for their similarity to a compendium of drugs whose effects on the yeast collection have been previously reported.We found that the effects of spaceflight have high concordance with the effects of DNA-damaging agents and changes in redox state, suggesting mechanisms by which spaceflight may negatively affect cell fitness.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, BC, Canada V6T 1Z3.

ABSTRACT
Spaceflight is a unique environment with profound effects on biological systems including tissue redistribution and musculoskeletal stresses. However, the more subtle biological effects of spaceflight on cells and organisms are difficult to measure in a systematic, unbiased manner. Here we test the utility of the molecularly barcoded yeast deletion collection to provide a quantitative assessment of the effects of microgravity on a model organism. We developed robust hardware to screen, in parallel, the complete collection of ~4800 homozygous and ~5900 heterozygous (including ~1100 single-copy deletions of essential genes) yeast deletion strains, each carrying unique DNA that acts as strain identifiers. We compared strain fitness for the homozygous and heterozygous yeast deletion collections grown in spaceflight and ground, as well as plus and minus hyperosmolar sodium chloride, providing a second additive stressor. The genome-wide sensitivity profiles obtained from these treatments were then queried for their similarity to a compendium of drugs whose effects on the yeast collection have been previously reported. We found that the effects of spaceflight have high concordance with the effects of DNA-damaging agents and changes in redox state, suggesting mechanisms by which spaceflight may negatively affect cell fitness.

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Related in: MedlinePlus

The Opticell Processing Module (OPM) designed for propagation of each deletion collection for ~21 generations of growth. The OPM comprises three commercially available optically clear chambers (Opticells, Nunc) that are joined by a manifold and scaffold that can be autoclaved and assembled rapidly. The manifold contains a multiway valve unit which mates to each Opticell or to an the off position using O-ring seals. The opposite side of the valve contains a Luer fitting into which a standard 3cc syringe is attached. To perform a growth assay in the OPM, each of the three chambers is filled with 7 mL of sterile growth media. Deletion pools are loaded into the inoculation syringe and then injected into Chamber A of the OPM, precooled to 4°C. Growth is initiated by warming the unit to 30°C. After 16–24 h, 0.5 mL is removed from Chamber A and injected and mixed into Chamber B using the same syringe. This is repeated to continue multigenerational growth in Chamber C.
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fig1: The Opticell Processing Module (OPM) designed for propagation of each deletion collection for ~21 generations of growth. The OPM comprises three commercially available optically clear chambers (Opticells, Nunc) that are joined by a manifold and scaffold that can be autoclaved and assembled rapidly. The manifold contains a multiway valve unit which mates to each Opticell or to an the off position using O-ring seals. The opposite side of the valve contains a Luer fitting into which a standard 3cc syringe is attached. To perform a growth assay in the OPM, each of the three chambers is filled with 7 mL of sterile growth media. Deletion pools are loaded into the inoculation syringe and then injected into Chamber A of the OPM, precooled to 4°C. Growth is initiated by warming the unit to 30°C. After 16–24 h, 0.5 mL is removed from Chamber A and injected and mixed into Chamber B using the same syringe. This is repeated to continue multigenerational growth in Chamber C.

Mentions: In this study, we designed the Opticell Processing Module or OPM (Figure 1) that was capable of maintaining the yeast deletion collection as a pool grown in liquid culture for at least 20 generations in microgravity. The hardware comprised a liquid-sealed system of growth chambers (Opticells) that allowed for gas exchange across polystyrene membranes. Each OPM consisted of three Nunc Opticells held together with a common manifold and valve system that is autoclaved and attached with watertight O-ring seals. A 3 mL syringe connected to the manifold with a Luer fitting is used to transfer liquid between chambers and mix without breaking sterility and with minimal operator intervention. The valve on the manifold has four settings that connect the syringe to the following port locations 1: Off position, 2: Opticell A, 3: Opticell B, or 4: Opticell C. The OPM allows propagation of each deletion collection for a combined ~21 generations of growth when three chambers are used and the inoculum and transfer volumes are 0.5 mL.


Genes required for survival in microgravity revealed by genome-wide yeast deletion collections cultured during spaceflight.

Nislow C, Lee AY, Allen PL, Giaever G, Smith A, Gebbia M, Stodieck LS, Hammond JS, Birdsall HH, Hammond TG - Biomed Res Int (2015)

The Opticell Processing Module (OPM) designed for propagation of each deletion collection for ~21 generations of growth. The OPM comprises three commercially available optically clear chambers (Opticells, Nunc) that are joined by a manifold and scaffold that can be autoclaved and assembled rapidly. The manifold contains a multiway valve unit which mates to each Opticell or to an the off position using O-ring seals. The opposite side of the valve contains a Luer fitting into which a standard 3cc syringe is attached. To perform a growth assay in the OPM, each of the three chambers is filled with 7 mL of sterile growth media. Deletion pools are loaded into the inoculation syringe and then injected into Chamber A of the OPM, precooled to 4°C. Growth is initiated by warming the unit to 30°C. After 16–24 h, 0.5 mL is removed from Chamber A and injected and mixed into Chamber B using the same syringe. This is repeated to continue multigenerational growth in Chamber C.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4309212&req=5

fig1: The Opticell Processing Module (OPM) designed for propagation of each deletion collection for ~21 generations of growth. The OPM comprises three commercially available optically clear chambers (Opticells, Nunc) that are joined by a manifold and scaffold that can be autoclaved and assembled rapidly. The manifold contains a multiway valve unit which mates to each Opticell or to an the off position using O-ring seals. The opposite side of the valve contains a Luer fitting into which a standard 3cc syringe is attached. To perform a growth assay in the OPM, each of the three chambers is filled with 7 mL of sterile growth media. Deletion pools are loaded into the inoculation syringe and then injected into Chamber A of the OPM, precooled to 4°C. Growth is initiated by warming the unit to 30°C. After 16–24 h, 0.5 mL is removed from Chamber A and injected and mixed into Chamber B using the same syringe. This is repeated to continue multigenerational growth in Chamber C.
Mentions: In this study, we designed the Opticell Processing Module or OPM (Figure 1) that was capable of maintaining the yeast deletion collection as a pool grown in liquid culture for at least 20 generations in microgravity. The hardware comprised a liquid-sealed system of growth chambers (Opticells) that allowed for gas exchange across polystyrene membranes. Each OPM consisted of three Nunc Opticells held together with a common manifold and valve system that is autoclaved and attached with watertight O-ring seals. A 3 mL syringe connected to the manifold with a Luer fitting is used to transfer liquid between chambers and mix without breaking sterility and with minimal operator intervention. The valve on the manifold has four settings that connect the syringe to the following port locations 1: Off position, 2: Opticell A, 3: Opticell B, or 4: Opticell C. The OPM allows propagation of each deletion collection for a combined ~21 generations of growth when three chambers are used and the inoculum and transfer volumes are 0.5 mL.

Bottom Line: Spaceflight is a unique environment with profound effects on biological systems including tissue redistribution and musculoskeletal stresses.The genome-wide sensitivity profiles obtained from these treatments were then queried for their similarity to a compendium of drugs whose effects on the yeast collection have been previously reported.We found that the effects of spaceflight have high concordance with the effects of DNA-damaging agents and changes in redox state, suggesting mechanisms by which spaceflight may negatively affect cell fitness.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, BC, Canada V6T 1Z3.

ABSTRACT
Spaceflight is a unique environment with profound effects on biological systems including tissue redistribution and musculoskeletal stresses. However, the more subtle biological effects of spaceflight on cells and organisms are difficult to measure in a systematic, unbiased manner. Here we test the utility of the molecularly barcoded yeast deletion collection to provide a quantitative assessment of the effects of microgravity on a model organism. We developed robust hardware to screen, in parallel, the complete collection of ~4800 homozygous and ~5900 heterozygous (including ~1100 single-copy deletions of essential genes) yeast deletion strains, each carrying unique DNA that acts as strain identifiers. We compared strain fitness for the homozygous and heterozygous yeast deletion collections grown in spaceflight and ground, as well as plus and minus hyperosmolar sodium chloride, providing a second additive stressor. The genome-wide sensitivity profiles obtained from these treatments were then queried for their similarity to a compendium of drugs whose effects on the yeast collection have been previously reported. We found that the effects of spaceflight have high concordance with the effects of DNA-damaging agents and changes in redox state, suggesting mechanisms by which spaceflight may negatively affect cell fitness.

Show MeSH
Related in: MedlinePlus