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The objectives of the study are to determine the kinetics of production and to characterize the unique aspects of genetic and developmental lesion induced in animal cells by radiation present in the space environment. Special attention is given to heavy charged particles. The organism Caenorhabditis elegans, a simple nematode, is used as a model system for a coordinated set of ground-based and flight experiments.
This volume includes papers on correlations between aging effects and space effects on biosystems, with particular attention given to the effects on the cardiovascular system, bone, sleep, cellular systems, immunological system, and genetics. Papers are presented on NASA and NIA plans and opportunities, the age effect on the posture and circulation, the cardiovascular physiology in space flight, and age-related bone changes. Attention is given to research on sleep, circulation rhythms, and aging and its applications to manned spaceflight; sleep and circadian rhythms; altered cell function in microgravity; and the heterogeneity of changes in lymphoproliferative ability with increasing age. Also included is a review of cellular immunosenescence, a paper on the immune response during space flight, and a paper on Caenorhabditis elegans as a model system for space biology studies.
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. Flight sample mutation rate was approximately 8-fold higher than ground controls which exhibited laboratory spontaneous frequencies.
In order to estimate radiation exposure in space, experiments were conducted during the 1st International Microgravity Laboratory (IML-1) mission in order to isolate genetic changes in animal cells caused by cosmic rays. The space measurements were evaluated against results from synthetic cosmic rays produced by particle accelerators on the ground. The biological material used was the tiny soil nematode, Caenorhabditis elegans. The measurements were made by thermoluminescent detectors and plastic nuclear track detectors. The development and the chromosome mechanics in microgravity were studied, and the mutagenesis induced by radiation exposure was analyzed. The results showed that there are no obvious differences in the development, behavior and chromosome mechanics, as a function of gravity unloading (reproduction, self-fertilization and mating of males with hermaphrodites, gross anatomy, symmetry and gametogenesis, pairing, disjoining and recombination of chromosomes). A variety of mutants were isolated, and it was noted that mutants isolated from regions of identified high particles were more severely affected than those isolated by random screening. Linear energy transfer particles seem to favor large scale genetic lesions.
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, Diamidinophenolindole (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.
The central dogma of molecular biology states that information is stored in DNA, transcribed to messenger RNA (mRNA) and then translated into proteins. This picture is significantly augmentated when we consider the action of certain proteins in regulating transcription. These transcription factors provide a feedback pathway by which genes can regulate one another's expression as mRNA and then as protein. To review: DNA, RNA and proteins have different functions. DNA is the molecular storehouse of genetic information. When cells divide, the DNA is replicated, so that each daughter cell maintains the same genetic information as the mother cell. RNA acts as a go-between from DNA to proteins. Only a single copy of DNA is present, but multiple copies of the same piece of RNA may be present, allowing cells to make huge amounts of protein. In eukaryotes (organisms with a nucleus), DNA is found in the nucleus only. RNA is copied in the nucleus then translocates(moves) outside the nucleus, where it is transcribed into proteins. Along the way, the RNA may be spliced, i.e., may have pieces cut out. RNA then attaches to ribosomes and is translated to proteins. Proteins are the machinery of the cell other than DNA and RNA, all the complex molecules of the cell are proteins. Proteins are specialized machines, each of which fulfills its own task, which may be transporting oxygen, catalyzing reactions, or responding to extracellular signals, just to name a few. One of the more interesting functions a protein may have is binding directly or indirectly to DNA to perform transcriptional regulation, thus forming a closed feedback loop of gene regulation. The structure of DNA and the central dogma were understood in the 50s; in the early 80s it became possible to make arbitrary modifications to DNA and use cellular machinery to transcribe and translate the resulting genes; more recently, genomes (i.e., the complete DNA sequence) of many organisms have been sequenced. This large-scale sequencing began with simple organisms, viruses and bacteria, progressed to eukaryotes such as yeast, and more recently (1998) progressed to a multi-cellular animal, the nematode Caenorhabditis elegans. Sequencers have now moved on to the fruit fly Drosophila melanogaster, whose sequence is slated for completion by the end of 1999. The human genome project is expected to determine the complete sequence of all 3 billion bases of human DNA within the next five years. In the wake of genome-scale sequencing, further instrumentation is being developed to assay gene expression and function on a comparably large scale. Much of the work in computational biology focuses on computational tools used in sequencing, finding genes that are related to a particular gene, finding which parts of the DNA code for proteins and which do not, understanding what proteins will be formed from a given length of DNA, predicting how the proteins will fold from a one-dimensional structure into a three dimensional structure, and so on. Much less computational work has been done regarding the function of proteins. One reason for this is that different proteins function very differently, and so work on protein function is very specific to certain classes of proteins. There are, for example, proteins such enzymes that catalyze various intracellular reactions, receptors that respond to extracellular signals and ion channels that regulate the flow of charged particles into and out of the cell. In this chapter, we will consider a particular class of proteins called transcription factors(TFs), which are responsible for regulating when a certain gene is expressed in a certain cell, which cells it is express in, and how much is expressed. Understanding these processes will involve developing a deeper understanding of transcription, translation, and the cellular processes that control those processes. All of these elements fall under the aegis of gene regulation or more narrowly transcriptional regulation. Some of the key questions in gene regulation are: What genes are expressed in a certain cell at a certain time? How does gene expression differ from cell to cell in a multicellular organism? Which proteins act as transcription factors, i.e., are important in regulating gene expression? From questions like these, we hope to understand which genes are important for various macroscopic processes. Nearly all of the cells of a multicellular organism contain the same DNA. Yet this same genetic information yields a large number of different cell types. The fundamental difference between a neuron and a liver cell, for example, is which genes are expressed. Thus understanding gene regulation is an important step in understanding development. Furthermore, understanding the usual genes that are expressed in cells may give important clues about various diseases. Some diseases, such as sickle cell anemia and cystic fibrosis, are caused by defects in single, non-regulatory genes; others, such as certain cancers, are caused when the cellular control circuitry malfunctions - an understanding of these diseases will involve pathways of multiple interacting gene products. There are numerous challenges in the area of understanding and modeling gene regulation. First and foremost, biologists would like to develop a deeper understanding of the processes involved, including which genes and families of genes are important, how they interact, etc. From a computation point of view, there has been embarrassingly little work done. In this chapter there are many areas in which we can phrase meaningful, non-trivial computational questions, but questions that have not been addressed. Some of these are purely computational (what is a good algorithm for dealing with a model of type X) and others are more mathematical (given a system with certain characteristics, what sort of model can one use? How does one find biochemical parameters from system-level behavior using as few experiments as possible?). In addition to biological and algorithmic problems, there is also the ever-present issue of theoretical biology - what general principles can be derived from these systems, what can one do with models other than just simulate time-courses, what can be deduced about a class of systems without knowing all the details? The fundamental challenge to computationalists and theorists is to add value to the biology - to use models, modeling techniques and algorithms to understand the biology in new ways.
The Forkhead or Fox gene family encodes putative transcription factors. There are at least four Fox genes in yeast, 16 in Drosophila melanogaster (Dm) and 42 in humans. Recently, vertebrate Fox genes have been classified into 17 groups named FoxA to FoxQ. Here, we extend this analysis to invertebrates, using available sequences from D. melanogaster, Anopheles gambiae (Ag), Caenorhabditis elegans (Ce), the sea squirt Ciona intestinalis (Ci) and amphioxus Branchiostoma floridae (Bf), from which we also cloned several Fox genes. Phylogenetic analyses lend support to the previous overall subclassification of vertebrate genes, but suggest that four subclasses (FoxJ, L, N and Q) could be further subdivided to reflect their relationships to invertebrate genes. We were unable to identify orthologs of Fox subclasses E, H, I, J, M and Q1 in D. melanogaster, A. gambiae or C. elegans, suggesting either considerable loss in ecdysozoans or the evolution of these subclasses in the deuterostome lineage. Our analyses suggest that the common ancestor of protostomes and deuterostomes had a minimum complement of 14 Fox genes.
Ctenophores are thoroughly modern animals whose ancestors are derived from a separate evolutionary branch than that of other eumetazoans. Their major longitudinal body axis is the oral-aboral axis. An apical sense organ, called the apical organ, is located at the aboral pole and contains a highly innervated statocyst and photodetecting cells. The apical organ integrates sensory information and controls the locomotory apparatus of ctenophores, the eight longitudinal rows of ctene/comb plates. In an effort to understand the developmental and evolutionary organization of axial properties of ctenophores we have isolated a forkhead gene from the Brain Factor 1 (BF-1) family. This gene, ctenoBF-1, is the first full-length nuclear gene reported from ctenophores. This makes ctenophores the most basal metazoan (to date) known to express definitive forkhead class transcription factors. Orthologs of BF-1 in vertebrates, Drosophila, and Caenorhabditis elegans are expressed in anterior neural structures. Surprisingly, in situ hybridizations with ctenoBF-1 antisense riboprobes show that this gene is not expressed in the apical organ of ctenophores. CtenoBF-1 is expressed prior to first cleavage. Transcripts become localized to the aboral pole by the 8-cell stage and are inherited by ectodermal micromeres generated from this region at the 16- and 32-cell stages. Expression in subsets of these cells persists and is seen around the edge of the blastopore (presumptive mouth) and in distinct ectodermal regions along the tentacular poles. Following gastrulation, stomodeal expression begins to fade and intense staining becomes restricted to two distinct domains in each tentacular feeding apparatus. We suggest that the apical organ is not homologous to the brain of bilaterians but that the oral pole of ctenophores corresponds to the anterior pole of bilaterian animals.
Gravitropism allows plant organs to direct their growth at a specific angle from the gravity vector, promoting upward growth for shoots and downward growth for roots. Little is known about the mechanisms underlying gravitropic signal transduction. We found that mutations in the ARG1 locus of Arabidopsis thaliana alter root and hypocotyl gravitropism without affecting phototropism, root growth responses to phytohormones or inhibitors of auxin transport, or starch accumulation. The positional cloning of ARG1 revealed a DnaJ-like protein containing a coiled-coil region homologous to coiled coils found in cytoskeleton-interacting proteins. These data suggest that ARG1 participates in a gravity-signaling process involving the cytoskeleton. A combination of Northern blot studies and analysis of ARG1-GUS fusion-reporter expression in transgenic plants demonstrated that ARG1 is expressed in all organs. Ubiquitous ARG1 expression in Arabidopsis and the identification of an ortholog in Caenorhabditis elegans suggest that ARG1 is involved in other essential processes.
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 plactic nuclear track detectors to correlate fluence of HZE particles with genetic events.
Genetic transformation with in vivo reporter genes for fluorescent proteins can be performed on a variety of organisms to address fundamental biological questions. Model organisms that may utilize an ISS imager include unicellular organisms (Saccharomyces cerevisiae), plants (Arabidopsis thaliana), and invertebrates (Caenorhabditis elegans).
Genetic transformation with in vivo reporter genes for fluorescent proteins can be performed on a variety of organisms to address fundamental biological questions. Model organisms that may utilize an ISS imager include unicellular organisms (Saccharomyces cerevisiae), plants (Arabidopsis thaliana), and invertebrates (Caenorhabditis elegans). The multispectral fluorescence imager (MSFI) will have the capability to accommodate 10 cm x 10 cm Petri plates, various sized multi-well culture plates, and other custom culture containers. Features will include programmable temperature and light cycles, ethylene scrubbing (less than 25 ppb), CO2 control (between 400 ppm and ISS-ambient levels in units of 100 ppm) and sufficient airflow to prevent condensation that would interfere with imaging.
The Biological Research in Canisters - LED (BRIC-LED) is a biological research system that is being designed to complement the capabilities of the existing BRIC-Petri Dish Fixation Unit (PDFU) for the Space Life and Physical Sciences (SLPS) Program. A diverse range of organisms can be supported, including plant seedlings, callus cultures, Caenorhabditis elegans, microbes, and others. In the event of a launch scrub, the entire assembly can be replaced with an identical back-up unit containing freshly loaded specimens.
Spectrum is a multispectral fluorescence imager designed for capturing in vivo genetic expression in a variety of biological organisms, providing a capability that does not currently exist on the International Space Station (ISS). Researching organisms that have been transformed with in vivo reporter genes ligated with fluorescent proteins allows the scientific community to further understand the fundamental biological responses of these organisms when subjected to space environments. Model organisms that may utilize multispectral imaging on the ISS include unicellular organisms (e.g. Saccharomyces cerevisiae), plants (e.g. Arabidopsis thaliana), and invertebrates (e.g. Caenorhabditis elegans).
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.
This oral presentation described the effects of cosmic radiation and microgravity on the stability of the genome and the fidelity of development in the small nematode, Caenorhabditis elegans.
During meiotic prophase, the essential events of homolog pairing, synapsis, and recombination are coordinated with meiotic progression to promote fidelity and prevent aneuploidy. The conserved AAA+ ATPase PCH-2 coordinates these events to guarantee crossover assurance and accurate chromosome segregation. How PCH-2 accomplishes this coordination is poorly understood. Here, we provide evidence that PCH-2 decelerates pairing, synapsis and recombination in C. elegans by remodeling meiotic HORMADs. We propose that PCH-2 converts the closed versions of these proteins, which drive these meiotic prophase events, to unbuckled conformations, destabilizing interhomolog interactions and delaying meiotic progression. Further, we find that PCH-2 distributes this regulation among three essential meiotic HORMADs in C. elegans: PCH-2 acts through HTP-3 to regulate pairing and synapsis, HIM-3 to promote crossover assurance, and HTP-1 to control meiotic progression. In addition to identifying a molecular mechanism for how PCH-2 regulates interhomolog interactions, our results provide a possible explanation for the expansion of the meiotic HORMAD family as a conserved evolutionary feature of meiosis. Taken together, our work demonstrates that PCH-2’s remodeling of meiotic HORMADs has functional consequences for the rate and fidelity of homolog pairing, synapsis, recombination and meiotic progression, ensuring accurate meiotic chromosome segregation.
The Loma Linda University (LLU) Radiobiology Program coordinates basic research and proton beam service activities for the university and extramural communities. The current focus of the program is on the biological and physical properties of protons and the operation of radiobiology facilities for NASA-sponsored projects. The current accelerator, supporting facilities and operations are described along with a brief review of extramural research projects supported by the program. These include space craft electronic parts and shielding testing as well as tumorigenesis and animal behavior experiments. An overview of research projects currently underway at LLU is also described. These include: 1) acute responses of the C57Bl/6 mouse immune system, 2) modulation of gene expression in the nematode C. elegans and rat thyroid cells, 3) quantitation of dose tolerance in rat CNS microvasculature, 4) behavioral screening of whole body proton and iron ion-irradiated C57Bl/6 mice, and 5) investigation of the role of cell integration into epithelial structures on responses to radiation.