Signs of Life A Report Based on the April 2000 Workshop on Life Detection Techniques (2002) / Chapter Skim
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Workshop Papers Session 3: Detecting Extant Life
Workshop Papers Session 3: Detecting Extant Life
Pages 103-146
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From page 103... ...
Session 3: Detecting Extant Life 103
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From page 105... ...
· Electron energy loss spectroscopy allows one to look at chemical binding states of 100-nm thin section specimens, with about 0.5-eV energy resolution in most instruments and a spatial resolution of 5 nm or better. · X-ray microscopy allows one to look at somewhat thicker sections, with an improved energy resolution of about 0.1 eV and a spatial resolution of 30-50 nm.
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From page 106... ...
More importantly, soft x-rays are well suited to providing high spatial resolution images of organic specimens. By operating at photon energies above ~289 eV, one has enough energy to remove inner shell electrons from carbon atoms by ionization.
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From page 107... ...
Zimba, "Soft X-ray Spectroscopy from Image Sequences with Sub-100 nm Spatial Resolution," Journal Microsc.
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From page 108... ...
Distinctive etch patterns of mineral surfaces may comprise mineralogical biosignatures within Zones 1, 2, and 3. Sample preparation techniques designed to preserve the morphology, antigenicity, and mineralogy of these highly hydrated and complex samples for highresolution electron microscopy are an important tool for evaluating these materials.
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From page 109... ...
High-pressure cryofixed, freeze substituted zero loss energy-filtered transmission electron microscopy micrograph of ultramicrotomed thin section.
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Characterization of complex mixtures of nanocrystalline minerals, highly hydrated extracellular polymers of variable composition and structure, and a diverse microbial assemblage is required to understand microbe-mineral interactions leading to formation and preservation of mineralogical biosignatures. The size of the components and the scale of the heterogeneities dictate the use of high-resolution analytical scanning and transmission electron microscopy in combination with biological cytochemical methods.
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From page 111... ...
111 Given the aforementioned constraints imposed by geobiological samples, freeze substitution appears to be an extremely useful preparative method for mineralogical biosignatures research. Resultant epoxy-embedded samples are amenable to standard ultramicrotome.
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From page 112... ...
Therefore, this method may lend itself to general use with a broad spectrum of uncharacterized materials. We have shown that different types of DNA molecules can be discriminated based on the blockade level and duration they produce when forced to translocate through our pore.
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From page 113... ...
For each event, we measured the translocation duration, tT, and the normalized blockade level defined as r IB = (IEvent>/(IOpen) The fractional blockade level, IB, is defined as the ratio of the average current at the translocation period to the average open pore current.
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(a) Event diagram showing translocation duration versus blockade level for poly(dA)
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From page 115... ...
The ratio between the translocation duration times of different polymer types is also extended at lower temperatures, allowing easier discrimination between several polymer types in the same sample. Probing Foreign Groups on DNA Molecules As discussed earlier, the electrical current and translocation time measurements are expected to be sensitive to the bulkiness of the probed molecule.
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From page 116... ...
Typical current traces recorded from SO-mer DNA molecules each labeled with two bulk groups at positions 25 and 37 from the S'-end. In this experiment, more than 85 percent of the events showed a clear two dips in the current from the initial plateau at around 18 percent, corresponding to the additional blockades caused by the extra groups.
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From page 117... ...
Almost all Earth science departments have stable isotope ratio mass spectrometers, the necessary analytical equipment for undertaking studies in rocks, carbonate fossils, or other geological materials. In recent years, owing to the development and marketing of stable isotope instrumentation that can be largely automated, the technology is becoming an integral part of most ecological and biological studies.
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From page 118... ...
In contrast to the richness in studies of stable isotopes in living organisms, an inventory of the proteins and enzymes responsible for isotopic partitioning is at an early stage of a new pulse of active growth. Gel electrophoresis, the separation of molecules based on molecular weight and charge by applying an electrical field to a gel containing a mixture of proteins, has been the principal tool for protein biochemical studies.
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From page 119... ...
can be used to assemble a quick snapshot of extant life. While it is important to study the stable isotope compositions and fractionations in living organisms as tools for finding signatures of life on other planetary bodies, it is equally important to know whether biological isotopic signals survive on geological time scales.
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From page 120... ...
Once molecules are selectively bound to the surface of the chip, they are desorbed and analyzed by time-of-flight mass spectrometry, which is capable of resolving 1 dalton in molecular weight in a molecule that may be as large as 100,000 daltons. The molecular weights of molecules that are desorbed by laser energy are then stored digitally, and the patterns of their molecular weights can be compared to what is expected from genome analyses.
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From page 121... ...
On this graph, it can be seen that there are numerous molecules in the molecular weight region greater than 1,000 and less than 1,200. Peptidoglycan units, the backbones of microbial cell walls, have molecular weights in this range.
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From page 122... ...
The technology is changing rapidly as new innovations in nanotechnology are developed and incorporated. Future research in stable isotopes at the molecular level, coupled to molecular fingerprinting of biochemical molecules in the geological record, should provide a new level of biosignature for the search for extant or extinct life on other planets.
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Magnetotactic bacteria are the simplest organisms known to contain biogenic magnetite,62 63 a typical example of which is shown in Figure 1. They precipitate individual submicron-sized magnetite crystals within an
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From page 124... ...
These are "Nature's biological compasses," and natural selection has perfected the size, shape, composition, crystallographic orientation, and ultrastructure of individual crystals within the cells in order to enhance their magnetic properties.66 Chains of these magnetosomes act as simple compass needles that passively torque the bacterial cells into alignment with Earth's magnetic field and allow them to seek the microaerophilic zone at the mud-water interface of most natural aqueous environments. These bacteria swim to the magnetic north in the Northern Hemisphere,67 to the magnetic south in the Southern Hemisphere,68 69 and both ways on the geomagnetic equator.70~7~ Magnetosomes have also been found in eukaryotic magnetotactic algae, with each cell containing several thousand crystals.72 Figure 2 illustrates in schematic ~ Source ..
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Highresolution transmission electron microscopy (HRTEM) studies reveal that bacterial magnetites are nearly perfect crystals, which (2)
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From page 126... ...
magnetic field at the end of a magnetosome.79 As shown in Figure 4, bacterial magnetite crystals are restricted to a size range from 35 to about 500 nm, with shapes that confine them to the single-domain magnetic stability field.~° 8i Inorganic magnetites tend to have log-normal size distributions that often spill up into the multidomain size region; this arises from fundamental inorganic growth principles.82 Bacterial magnetites tend to be rather pure iron oxide, with no detectable titanium, chromium, or aluminum, which are often present in geologically produced magnetite. This is presumably due to the biopurification processes produced by the iron-specific binding and transport proteins shown in Figure 2.
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From page 127... ...
Although bacterial magnetite crystals produced by some common strains (e.g., equant cubo-octahedra crystals of Magnetosprillum magnetotacticum) fall outside the central area of the diagram, no inorganically produced population of magnetite crystals has yet been found that inhabits this central region.
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From page 128... ...
elongated morphology, which is rather peculiar for a cubic mineral. These properties are the end result of natural selection for magnetic properties of the magnetite crystals, and they are a unique biological fingerprint.
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From page 129... ...
mass spectrometers that have very high mass range and sensitivity. Mass spectrometers have been found in space applications where they have been used to measure atmospheric species and small organic compounds.
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From page 130... ...
The Time-of-flight Mass Spectrometer In the TOF mass spectrometer ions are formed in a short source region (Figure 1) , accelerated by the electric field formed by the voltage V to a constant energy and injected into a longer drift region (D)
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From page 131... ...
This approach, which they have termed phyloproteomics, is a robust technique for identification of organisms that does not depend upon reproducible growth conditions or mass spectra. One should expect that MALDI time-offlight mass spectrometers would provide similar biomarkers in both terrestrial and extraterrestrial scenarios, in which it would be possible to rapidly assess whether these are of known origin or are similar to known species.
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From page 132... ...
DNA is known to persist in some noncellular contexts, and DNA bound to surfaces such as clay is resistant to degradation.l26 However, other Copolymers such as RNA and phospholipids are much less stable and generally are rapidly degraded following cell death.l27 128 For example, reverse transcriptase polymerase chain reaction (RT-PCR) has been used to confirm that microbial populations detected at the ribosomal DNA level are metabolically active.
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in the adenylate pool. The energy charge of bacteria under normal conditions varies between 0.87 and 0.95 irrespective of growth rate.
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identification of Diagnostic Nucleic Acid Sequences in Environmental Samples samples: There are three basic methods now used to recover nucleic acid sequence information from environmental
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DNA probe hybridization; and 3. Restriction enzyme digestion.
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From page 136... ...
. Some examples of traits, and corresponding genes, sufficiently conserved to be easily identified in environmental samples include genes for nitrogen fixation,~65 Ni-Fe hydrogenate, CO2 fixation, and the dissimilatory sulfite reductase.~67 ~68 Other Technical Issues The following technical issues are key considerations in detection.
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From page 137... ...
The exploration of the diversity of life on Earth has revealed key genetic components that are conserved in all free-living organisms. The most conserved of these genes are the ribosomal RNA genes, one class of which is called the 16S or 18S RNA genes.~70 Ribosomal RNAs are the main structural and catalytic components of the ribosome, the molecular machine that translates RNA into proteins.
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From page 138... ...
If such microbial life exists on Mars currently and is more abundant than about one cell per gram at the landing site, PCR is sensitive enough to detect the signature of life amplification of DNA with particular DNA sequence features using these DNA primers. There are two conditions under which life on Mars should resemble life on Earth to the point of sharing ribosomal RNA sequences: exchange between Mars and Earth or, more generally, common ancestry from another source, perhaps even outside of the solar system.
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From page 139... ...
Samples would then be mixed with reagents including PCR primers corresponding to the universal elements of ribosomal genes and thermally cycled. Bona fide ribosomal RNA genes would be expected to generate a PCR product of a size in a narrow range.
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In principle, the DNA sequence analysis chip and charge coupled device electronics to read the chip scanner could be similar in size to a portable CD player. Such a micromicrobial analysis machine would also encourage useful advances in portable terrestrial DNA analyses as spinoffs.
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Zimba, "Soft X-ray Spectroscopy from Image Sequences with Sub-100 nm Spatial Resolution," Journal Microsc.
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From page 142... ...
J.L. Kirschvink, "Paleomagnetic Evidence for Fossil Biogenic Magnetite in Western Crete," Earth Planet.
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From page 143... ...
K.L. Thomas-Keprta et al., "Elongated Prismatic Magnetite Crystals in ALH84001 Carbonate Globules: Potential Martian Magnetofossils," Geochim.
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Z Gibbs-Eggar, '~Possible Evidence for Dissimilatory Bacterial Magnetite Dominating the Magnetic Properties of Recent Lake Sediments," Earth Planet.
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Woese, "A New Version of the RDP (Ribosomal Database Project) ," Nucleic Acids Research 27(1)
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From page 146... ...
Stolper, "Low-temperature Carbonate Concretions in the Martian Meteorite ALH84001: Evidence from Stable Isotopes and Mineralogy," Science 275:1633-1638, 1997.
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