Wayne W. Schubert

Radiation effects in Caenorhabditis elegans, mutagenesis by high and low LET ionizing radiation

The nematode C. elegans was used to measure the effectiveness of high-energy ionized particles in the induction of 3 types of genetic lesions. Recessive lethal mutations in a 40-map unit autosomal region, sterility, and X-chromosome nondisjunction or damage were investigated. Induction rates were measured as a function of linear energy transfer, LET infinity, for 9 ions of atomic number 1-57 accelerated at the BEVALAC accelerator. Linear kinetics were observed for all 3 types of lesions within the dose/fluence ranges tested and varied strongly as a function of particle LET infinity. Relative Biological Effectiveness (RBE) values of up to 4.2 were measured and action cross sections were calculated and compared to mutagenic responses in other systems.

Radiation effects in nematodes: Results from IML-1 experiments

The nematode Caenorhabditis elegans was exposed to natural space radiation using the ESA Biorack facility aboard Spacelab on International Microgravity Laboratory 1, STS-42. For the major experimental objective dormant animals were suspended in buffer or on agar or immobilized next to CR-39 plastic nuclear track detectors to correlate fluence of HZE particles with genetic events. This configuration was used to isolate mutations in a set of 350 essential genes as well as in the unc-22 structural gene. From flight samples 13 mutants in the unc-22 gene were isolated along with 53 lethal mutations from autosomal regions balanced by a translocation eT1(III;V). Preliminary analysis suggests that mutants from worms correlated with specific cosmic ray tracks may have a higher proportion of rearrangements than those isolated from tube cultures on a randomly sampled basis. Right sample mutation rate was approximately 8-fold higher than ground controls which exhibited laboratory spontaneous frequencies.

Development and chromosome mechanics in nematodes: Results from IML-1

A subset of the Caenorhabditis elegans nematodes flown aboard Biorack on IML-1 was analyzed for the fidelity of development and the mechanics of chromosomes at meiosis. To assess meiosis, mutant worms marked at two linked or unlinked loci were inoculated as heterozygous hermaphrodites and allowed to self fertilize. Mendelian segregation ratios and recombination frequency were measured for offspring produced at 1XG or in microgravity. To assess development, worms and embryos were fixed and stained with the DNA dye, DAPI, or antibodies specific for antigens expressed in germ cells, pharyngeal and body wall muscles, and gut cells. The distribution of cytoplasmic determinants, cell nuclei counts and positions were scored to assess symmetry relations and anatomical features.

Yeasts producing exopolysaccharides with drag-reducing activity

Abstract A search was conducted for yeast strains that could be cultured on methanol, ethanol, or glucose and that produce exopolysaccharides which reduce friction in turbulent liquid flows. Five yeast strains and one yeast-like fungus produced drag-reducing exopolysaccharides when grown on glucose and/or ethanol: Candida boidinii, Cryptococcus laurentii, Hansenula capsulata, Lipomyces starkeyii, Rhinocladellia elatior , and Rhodotorula glutinis . Polysaccharides with specific drag-reducing activities comparable to commercial xanthan gum were isolated from C. laurentii, R. glutinis , and R. elatior , but only when grown on glucose or ethanol. The exopolysaccharide from C. boidinii reduced drag when grown on methanol. A preliminary characterization of this previously unreported exopolysaccharide revealed that it is composed of glucose and mannose in an approximately 1:1 ratio. It is a relatively homogeneous polymer with a molecular weight of about 850 Kd and its production is favored by inclusion of ≥0.11% tryptone in the growth medium.

Determination of Lethality Rate Constants and D-Values for Heat-Resistant Bacillus Spores ATCC 29669 Exposed to Dry Heat from 125°C to 200°C

Exposing flight hardware to dry heat is a NASA-approved sterilization method for reducing microbial bioburden on spacecraft. The existing NASA specification only allows heating the flight hardware between 104°C and 125°C to reduce the number of viable microbes and bacterial spores. Also, the NASA specifications only allow a four log reduction by dry heat microbial reduction because very heat-resistant spores are presumed to exist in a diverse population (0.1%). The goal of this research was to obtain data at higher temperatures than 125°C for one of the most heat-resistant microorganisms discovered in a spacecraft assembly area. These data support expanding the NASA specifications to temperatures higher than 125°C and relaxing the four log reduction specification. Small stainless steel vessels with spores of the Bacillus strain ATCC 29669 were exposed to constant temperatures between 125°C and 200°C under both dry and ambient room humidity for set time durations. After exposures, the thermal spore exposure vessels were cooled and the remaining spores recovered and plated out. Survivor ratios, lethality rate constants, and D-values were determined at each temperature. The D-values for the spores exposed under dry humidity conditions were always found to be shorter than those under ambient humidity. The temperature dependence of the lethality rate constants was obtained by assuming that they obeyed Arrhenius behavior. The results are compared to those of B. atrophaeus ATCC 9372. In all cases, the D-values of ATCC 29669 are between 20 and 50 times longer than those of B. atrophaeus ATCC 9372.

Radiobiological studies with the nematode Caenorhabditis elegans. Genetic and developmental effects of high LET radiation

The biological effects of heavy charged particle (HZE) radiation are of particular interest to travellers and planners for long-duration space flights where exposure levels represent a potential health hazard. The unique feature of HZE radiation is the structured pattern of its energy deposition in targets. There are many consequences of this feature to biological endpoints when compared with effects of ionizing photons. Dose vs response and dose-rate kinetics may be modified, DNA and cellular repair systems may be altered in their abilities to cope with damage, and the qualitative features of damage may be unique for different ions. The nematode Caenorhabditis elegans is being used to address these and related questions associated with exposure to radiation. HZE-induced mutation, chromosome aberration, cell inactivation and altered organogenesis are discussed along with plans for radiobiological experiments in space.

Enhancement of carbohydrates in a methylotrophic yeast

Abstract The use of methylotrophic yeasts has been suggested for recycling CO 2 to human food in extended space missions. Since the human diet requires higher carbohydrate levels than those normally found in microbes, attempts were made to increase the levels of storage carbohydrates, principally glycogen, through cultural and genetic methods. The effect of defining cultural conditions for the methylotrophic yeast Hansenula polymorpha resulted in increasing the storage carbohydrate content of the dry weight of the cells from 30 to 46%. During this analysis, a growth requirement for potassium was discovered. Several mutant strains were selected for high glycogen storage on plates and analysed for storage carbohydrate levels in submerged culture. These strains exhibited an additional 4–16% increase in the levels of storage carbohydrates over the parent strain in stationary phase. One strain was also able to store excess carbohydrate during exponential growth at levels 10% above the parent strain. Through a combination of cultural control and genetic modification, carbohydrate levels in this yeast were raised to 60% of the cell dry weight. Through additional genetic selection these levels are likely to be increased even further.

Supercritical CO 2 Cleaning for planetary protection and contamination control

We have designed and built a new Supercritical CO2 Cleaning (SCC) system1,2 to conduct cleaning efficiency studies using Supercritical CO2 and liquid CO2 to remove trace amounts of microbial and organic contaminants from spacecraft material surfaces. The objective of this task is to develop an effective CO2 cleaning method and to demonstrate and validate its ability to achieve ultra-clean surfaces of sample handling devices, sample storage units, and science instruments. This new capability will meet planetary protection and contamination control requirements for future Astrobiology science missions. The initial cleaning test results using this new cleaning device showed that both supercritical CO2 and liquid CO2 could achieve cleanliness levels of 0.01 µg/cm2 or less for hydrophobic contaminants. Experiments under supercritical condition using compressed Martian air mix, which consists of 95% CO2, produced similar cleaning effectiveness on the hydrophobic compounds. This opens up the possibility of further development potential for in situ CO2 cleaning and sterilization using Martian air for future Mars missions. We plan to further investigate the cleaning condition for hydrophilic compounds and bacterial spores, as well as introducing polar co-solvent to the cleaning apparatus.

Certification of vapor phase hydrogen peroxide sterilization process for spacecraft application

This paper describes the selection process and research activities JPL is planning to conduct for certification of hydrogen peroxide as a NASA approved technique for sterilization of various spacecraft parts/components and entire modern spacecraft.

Evaluation of sample preservation methods for space missions

This paper will present the research data using samples collected from the Mars 2001 orbiter, Odyssey, and all environmental samples collected from the cleanroom, during final assembly.