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Technical University of Muenchen at Weihenstephan, Department of Botany, Freising, Germany
1Correspondence: A. Hahn, Technical University of Muenchen at Weihenstephan, Department of Botany, Alte Akademie 12, D-85350 Freising, Germany. E-mail: aeh{at}weihenstephan.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: fungal genetics • sexual recombination • radiation effects
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To study the reproduction of eukaryotic organisms under spaceflight conditions, we employed crosses of two fungal spore color mutant strains. In the FUNGUS experiment on the S/MM 05 space shuttle mission (STS-81) in January 1997, the classic genetic method of tetrad analysis was employed to gather information on the influence of space conditions on recombination in eukaryotic organisms by comparing the second-division segregation of the spore color genes lu and r2 in the ascomycete Sordaria macrospora Auersw. The mission duration of 10 days enabled a full reproduction cycle. In parallel, a ground experiment and a 1-g on-board centrifuge were employed as controls to differentiate between the factors of reduced gravity and space radiation.
The classic genetic method of tetrad analysis was developed by Esser
and Straub (1)
, based on the original work of Lindegren
(2)
and Beadle and Tatum (3)
. In brief, it
utilizes the distribution of spores within the asci (spore-containing
cells within the fruiting body) to derive information on gene
segregation. First-division segregation of the alleles during meiosis
leads to ascus halves with spores of the same color. A cross-over
between the gene locus and the centromere results in second-division
segregation and consequently in two different kinds of spores within
one ascus half. The probability of cross-over events increases with the
distance between gene locus and centromere. Cross-over frequencies can
therefore be used as measures for map distances or, vice versa,
comparison of gene distances under different conditions show an
influence of these experimental conditions on the number of cross-over
events. If the distances between two genes are to be estimated,
two-factor crosses must be carried out.
Fungi are well suited for the study of more than one generation in one
experiment due to their short life cycle and their ease of handling.
The simplicity of the system is one of the demands for space
experiments (4)
. The ascomycete used, Sordaria
macrospora Auersw., is a well-defined object of classical and
molecular fungal genetics. Zimmermann et al. (5)
exposed
spores of Sordaria fimicola over 69 months on a satellite
and found a considerable loss of germination ability after their
return. In addition, the effects of space on the segregation of spore
color genes was analyzed by unordered tetrad analysis of a one-factor
cross, indicating an increase in mutation rates and chromosome strand
breaks.
Only a small number of experiments have so far been conducted to study
the cytogenetic aspects of reproduction under these conditions over a
number of consecutive generations. In work done in the context of the
SpaceHab IML-1 mission (January 1992), Nelson et al. (6)
assessed the influence of spaceflight conditions on
Caenorhabditis elegans over two generations. Development of
the nematodes proceeded without any obvious defects in anatomy, growth,
mating behavior, gene segregation, and recombination. However, a
significantly enlarged number of mutations were observed in space
samples compared with ground controls and their appearance could be
matched with the local energy deposition by cosmic rays. Bruschi and
Esposito (7)
used strains of diploid yeast cells to
determine effects of spaceflight conditions on mitotic crossing-over
and found clues to differences between pre-recombinational lesions of
flight and ground cultures, which may be due to effects on the
chromosome structure, the DNA physiology, or the quality of space
radiation.
The influences of cosmic radiation can only be studied in space.
Biological effects of cosmic ray heavy ions have been summarized by
Horneck (8)
. The irradiation leads primarily to double
strand breaks of the DNA. We employed single-cell gel electrophoresis
(comet assays) to assess DNA damage to cells of S.
macrospora, thus examining the parental generation in addition to
the offspring.
FUNGUS permitted the determination of space radiation as well as
microgravity influences on the recombination of genes. The aspect of
reproduction is relevant in long-term space missions where more than
one generation of organisms will be involved (9)
. In the
experiment FUNGUS, we addressed the basic question of whether
microgravity and/or radiation had any influence on cytogenetic
processes such as chromosome mechanics.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, were employed in the experiment. They have
non-allelic defects in genes that are responsible for melanin
synthesis. These defects result in spore color aberrations. Mutant
r2 is fertile, the spore color is brown. Mutant
lu spd is sterile (due to the defect gene spd),
the spore color is yellow (due to the defect gene lu). The
defect of gene spd blocks the life cycle of mutant lu
spd and is compensated by crossing of the strains. The
heterokaryon can then complete its cycle and produce ascospores. Gene
spd does not appear in the analysis of spore colors.
Figure 1
schematically depicts the result of a cross between the two mutants.
The asci contain eight spores each in a linear order. They do not
change their position during spore development, i.e. during meiosis
yielding four nuclei and the subsequent mitotic division yielding eight
nuclei, followed by free cell formation and spore differentiation.
Consequently, the products of meiosis are submitted as ordered tetrads
of spore pairs.
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The following tetrad types are obtained: 1) parental (P) ditypes (i.e. A1, D1), which only contain nuclei with parental genetic combination (r2+lu yielding yellow spores and r2lu+ yielding light-brown spores); 2) non-parental (R) ditypes (i.e. A2, D2), which only contain recombinant nuclei (r2+lu+ yielding black spores and r2lu yielding white spores); 3) tetratypes (T; i.e. A3, B, C), which contain nuclei with all four possible genetic combinations.
Culture of S. macrospora
Biorack type I containers (10)
were prepared with
air holes. Each container held four small Petri dishes with nutrient
medium [(11)
12 g/l fructose; 2.2 g/l KNO3; 1
g/l KH2PO4; 0.5 g/l MgSO4 ·
7 H2O; 200 µg/l ZnSO4 ·
H2O; 4 µg/l biotin; 175 mg/l arginin; 35 g/l agar; pH
6.1). Vibration controls were only performed to ensure stability of the
equipment under space shuttle starting and landing conditions. Biostack
particle track detector foils (Kodak cellulose nitrate, cutoff LET
approximately 1300 MeV/g/cm2 H2O) were mounted
on each polystyrene Petri dish stack. Each Petri dish was inoculated
with the two strains of S. macrospora in a distance of
approximately 20 mm to time their contact to flight day 1. A total of
eight containers were kept at microgravity
(µG2)and four containers were kept on the 1 g reference
centrifuge in the Biorack incubator INC-A [22°C (10)
].
Together with the ground parallels, this resulted in four sample
batches with different growth conditions: flight µG, flight 1
g, ground 1 g, and ground 1.4 g
(centrifuge control).
The fungal cultures and the nutrient medium were then stored on the ground at 4°C before the experiment was initiated in orbit by transfer to INC-A.
In orbit, the experiment consisted of the transfer of the 12 containers from the passive thermal conditioning units (PTCU, 4°C) to INC-A, telemetering images to ground on mission day 5 to check the growth of the cultures and transfer back to the PTCU for landing. On mission day 3, containers FUNGUS 9 and 10 had to be transferred to a 0.1-g environment for 1.48 h. At this time, however, both strains were already in contact and protoperithecia formed. The cultures had already passed the stage of meiotic cell divisions in their developmental cycle.
Microscopic evaluations
After the mission, ripe perithecia (fruiting bodies) were
isolated from the cultures and the ascospores within the asci were
examined microscopically (Axioplan, Zeiss, Oberkochen, Germany). The
perithecia were crushed under a microscope coverslip, causing them to
open and to display the asci (approximately 30 per perithecium) for
observation. The tetrad type of each ascus was then determined and
registered. The distribution of the tetrad types was analyzed for
fruiting bodies within 5 x 5-mm grids (approximately three to
four fruiting bodies per grid). The post-reduction frequency for each
spore color gene was thus determined for each grid.
The stacked foils from the particle track detectors were etched in 6 N NaOH and evaluated using a stereo macroscope (M400, Wild, Heerbrugg). Matching tracks in all three consecutive foils of one stack were considered to originate from an HZE particle trajectory. The impact zone of the HZE particles on the plane of the fungal hyphae and perithecia was determined from the particle trajectories and related to the 5 x 5-mm grid. A higher resolution than 5 x 5 mm was not attempted.
The mycelium of the samples was shaved off with a lancet and stored at
4°C under N-lauroylsarcosine (1% in 0.5 M EDTA) until
analysis to avoid post-mission repair (12)
. Single-cell
gel electrophoresis (comet) assays were performed by embedding the
hyphae in 0.8% low-melting agarose on microscope slides and subjecting
them to cell lysis in alkaline buffer (0.3 M NaOH, 0.03 M EDTA, 0.1%
sodium dodecyl sulfate, pH > 13.5). The slides were transferred
to 0.5 TBE buffer (50 mM Tris-boric acid, 10 mM EDTA, pH 8.0),
subjected to electrophoresis (1.2 V/cm, 10 min), and finally stained
with ethidium bromide. The migration distance of the DNA from the
center of the nucleus was visualized and measured manually on a laser
scanning microscope (CLSM, Leitz, Wetzlar, Germany) equipped with a
5-mW argon laser (488/514 nm). The alkaline lysis step in the assay
procedure transforms the radiation-induced single-strand breaks and
alkali-labile sites into double-strand breaks. Increasing DNA damage is
then detected as an increase in the migration distance of the DNA
fragments from the nucleus. The samples were observed microscopically
after electrophoresis and the migration distance of DNA from the center
of the nucleus was measured as tail length. A minimum of 100 nuclei
were evaluated in each sample batch and the mean migration
distance ± SD were used as an indicator for DNA
damage according to Fairbairn et al. (13)
.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Tetrad analysis
The distribution of tetrad types was examined microscopically.
Post-reduction frequencies were calculated for each spore color gene
and the genetic distance between the genes was derived from these data.
The results of these calculations are summarized in Table 1
.
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These figures show no difference in the post-reduction frequencies of the two genes and consequently in the genetic distance of the two genes lu and r2.
Statistical significance
The 
2 test is suitable to detect deviations in the
tetrad distribution in a sample from an expected distribution
(14)
. We used it here to examine deviations of the
distribution in the space samples from those in the ground samples. A
2 value of >12.51 for 6 degrees of freedom (7 possible
ascus types) indicates a significant deviation with an error
probability of <5%. The results of these calculations are summarized
in Table 2
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There is no significant difference between the sample batches at µG
and 1 g from space or between 1 g and 1.4
g in the ground control. This proves that there is no
influence of gravity on the recombination frequency of the two genes.
However, significant differences are found between the tetrad
distributions in space and on the ground, both in the overall
distribution (2 = 249.18) and in the 1-g
samples (2 = 55.23). This indicates that the
post-reduction frequencies of the two genes were influenced by
conditions in space. The 2 values yield more information
than the calculation of the post-reduction frequencies because
distributions are examined instead of averages.
Deviations of tetrad type distribution within each sample batch
Tetrad distribution was then considered separately for each 5 x 5-mm grid and from these data the post-reduction frequency of gene
lu was calculated for each grid. This was not possible for
gene r2, which is located too close to the
centromere. A statistically significant calculation of its
post-reduction frequency and consequently for the genetic distance of
the two genes is only possible for large sample numbers. For gene
lu, the calculated post-reduction frequency for each grid
was compared to the frequency calculated from the overall distribution
in the space samples with the use of the 2 test. The
results are shown in Table 3
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No fruiting body in the ground samples showed a significant deviation of tetrad type distribution from the overall distribution in the sample batch. In all spaceflight samples (µG and 1 g), however, approximately 15% of the fruiting bodies showed a significant deviation from the overall distribution. These deviations were overlooked when post-reduction frequencies were calculated for a complete sample batch but became apparent when each fruiting body was examined separately. Because they appeared at the same rate in the 1-g samples from the on-board centrifuge as well as in the µG samples, they cannot be caused by µG.
Localization of HZE particle tracks
The radiation dose rates during S/MM-05 were in total less than 10
mGy for the 10 days of the mission (15)
. The radiation
sensitivity of the tetrad analysis of
r2+lu x
r2lu+ was approximated to
around 500 mGy by a comparison with samples treated with heavy ions and
X-rays (data not shown). The observed increase in post-reduction
frequency of gene lu can therefore not be due to the
-component of space radiation.
The heavy-ion component of cosmic radiation was analyzed by evaluating
the etch-cone detector foils and relating the particle tracks to the
5 x 5-mm grid on the fungal cultures. Between 57 and 78 tracks
were detected per container; this corresponds to an HZE particle
density of 2.1/cm2 or 0.44 per 5 x 5-mm grid. Each of
the nine grids showing an increase in the recombination frequency of
gene lu was hit by at least one HZE particle (see Table 3
for all flight samples). Of the non-hit perithecia, a significant
increase in the post-reduction frequency of lu was obtained
in 0 of 40 cases. Of the 27 situations when an HZE track was present,
one-third (9)
showed a significant increase in the
post-reduction frequency of lu, this mirrors the fact that
the sexual recombination of the genes was completed after flight day 2.
It is consequently not possible to inversely deduce an increase of
crossing-over events from a particle impact, since the dosimeter foils
were present in the containers during the full 10 days of the mission.
All particle impacts were scored even after the sensitive phase of the
experiment, which was the forming of protoperithecia on flight day 2.
Ground experiments at the UNILAC heavy ion accelerator (Darmstadt, Germany) had previously demonstrated that heavy ions do indeed significantly increase the post-reduction frequency of this gene (data not shown).
Strand breaks in flight samples
The flight samples were analyzed for DNA damage in the hyphal
nuclei of the parental cells by employing the comet assay method. The
principle of the assay is based on the increasing migration distance of
DNA strands with increasing fragmentation. It is illustrated in
Figure 2
for a flight sample (Fig. 2a
) and for a nucleus of a cell
treated with X-rays at a dose of approximately 2 Gy (Fig. 2b
). The mean tail length for all four sample batches from
FUNGUS is shown together with the standard deviation in Table 4
.
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DNA damage in the flight samples was not increased when compared
to the ground controls. To study repair kinetics in S.
macrospora, cultures were irradiated with X-rays at a dose of
approximately 2 Gy and then allowed to repair for different times
before being subjected to evaluation by the comet assay. The results of
these tests are shown in Figure 3
.
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Most of the considerable DNA damage caused by radiation is repaired within 60 min at room temperature. The speed of these repair processes explains the absence of detectable DNA damage in the flight samples.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Tetrad analysis of the overall results of crosses of S.
macrospora spore color mutants revealed no significant differences
between crossing-over frequencies in space during S/MM-05 and
corresponding ground samples. However, when fruiting bodies were
examined individually, a small number of perithecia showed a remarkable
and significant increase in crossing-over frequencies. The data were
correlated with the evaluation of local energy deposits being
determined by Biostack detectors and indicate a strong influence of HZE
particles on the cross-over frequency in S. macrospora. We
found 0.44 HZE tracks/cm2 of the detector material. This is
consistent with previous counts (16)
.
Despite the low fluxes of the heavy ions, they should be of major
concern to living organisms and samples in space due to their ionizing
power (17)
. The total absorbed dose in Grays per kilogram
of tissue of heavy ions accounts only to some µGy but doses up to 200
Gy occur in distances <1 µm around their trajectories. Such doses
are strong enough to induce mutations in biological material. Because
heavy ions act only in a restricted area within the range of a few
micrometers, it is necessary to detect each impact of an HZE particle
in relation to the sample material for the correlation of biological
effects with the HZE particle impact.
The effects induced by the impact of HZE particles have been studied
during several space missions. T4 bacteriophages growing in close
vicinity (about 1 mm2) to an HZE impact showed a 14-fold
increase in mutation frequency [ASTP mission (17)
].
Bacillus subtilis spores (Apollo 16 and 17) showed normal
germination rates, but the formation of the first mature vegetative
cell was significantly reduced in spores hit by an HZE particle
(18)
. Very close to an impact (4 µm) the spores were
inactivated. In seeds of Arabidopsis thaliana or
Nicotiana tabaccum the development was significantly
disturbed. A loss of germination, embryo lethality, total inactivation
(120 µm around the impact) of the shoot meristem, seedling
abnormalities like hypertrophy, deformations of the cotyledons,
hypocotyl and root, or chlorophyll deficiency were measured
(19
, 20
). Nevzgodina et al. (21)
detected in Lactuca sativa seeds hit by HZE particles
irreparable damage of the genetic apparatus as demonstrated by the high
frequency of multiple chromosomal aberrations. One seed of Zea
mays that received two hits by HZE particles developed a somatic
mutation: large yellow stripes in all leaves (22)
.
Unicellular systems are best suited for a clear particle-object
correlation and for the discrimination of hit and non-hit objects in
space radiation biology (4)
. In this regard, fungi are
well suited for genetic experiments under space conditions. FUNGUS was
originally conceived to examine the influence of gravity on the genetic
recombination frequency. Clinostat experiments with this system had
pointed toward an effect of gravity compensation. However, clinostats
imperfectly simulate lack of gravity. Due to the configuration of the
experiment, it was not possible to correlate HZE impact and cytogenetic
process more precisely than on a 5 x 5-mm grid. The results of
FUNGUS are, however, incorporated into the concept of the next
spaceflight experiments aiming for a higher resolution. The resolution
of the Biostack configuration can be enhanced by placing the biological
objects in close proximity to the detector foils. The range of
secondary electron effects around the tracks may then also be
estimated, which would translate into a dose estimate at individual
nuclei.
Tetrad analysis of S. macrospora can still be considered to be a very sensitive system to determine effects of radiation on chromosomal mechanics of eukaryotes. These post-flight assays of cytogenetic processes give a static picture of the conditions during a defined time period in flight, i.e. when meiotic divisions occur. The Biostack, in contrast, shows the total amount of HZE particles during the mission, whereas the comet assay, the third method of evaluation that was employed in our experiment, mirrors the momentary damage to nuclei, which will be repaired, unless the samples are fixed or repair-deficient mutants are employed. These latter approaches are planned for future experiments.
Along with other molecular genetic methods such as the triple-lux assay
(23)
, the comet assay is a very sensitive tool to
determine the genotoxic effects of environmental conditions. The manual
measurement of the DNA migration distance, tail length, is less
accurate than some damage parameters that can be determined through
commercially available image analysis methods. To make up for the lack
of an image analysis system in our laboratory, the described buffer
system was optimized for the parameter of tail length
(24)
. Together with the rather large sample number and the
high resolution of a laser scanning microscope (25)
,
reliable data acquisition for DNA damage assessment is still possible.
The assay is especially well-suited to study repair kinetics and mechanisms because DNA damage below lethal levels is detected and may easily be quantified. The local energy deposition of up to 200 Gy is likely to severely damage the DNA present in close vicinity to an HZE particle track. It remains to be determined whether this amount of damage to a fungal nucleus can be repaired and comet assay experiments with irradiated cells are currently being conducted toward this goal. In future experiments, a comet assay system is conceived, where the cells, growing in immediate vicinity of the Biostack foils, will be fixed at different times after exposure in orbit, thus enabling a very high spatial and temporal resolution. Under space conditions, a highly asymmetric distribution of damage events is expected, with occasional nuclei receiving a high dose in a population receiving a background level dose. This should lead to occasional long-tailed comets in a field of control value nuclei. The unique methodology of the comet assay is able to resolve this phenomenon if the sample size is large enough to sample an appropriate number of hit vs. non-hit cells.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication September 18, 1998.
Revision received January 19, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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