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Monte-Carlo Simulations of Heavy Ions Track Structures and Applications

In space, astronauts are exposed to protons, high ]energy heavy (HZE) ions that have a high charge (Z) and energy (E), and secondary radiation, including neutrons and recoil nuclei produced by nuclear reactions in spacecraft walls or in tissue. The astronauts can only be partly shielded from these particles. Therefore, on travelling to Mars, it is estimated that every cell nucleus in an astronaut fs body would be hit by a proton or secondary electron (e.g., electrons of the target atoms ionized by the HZE ion) every few days and by an HZE ion about once a month. The risks related to these heavy ions are not well known and of concern for long duration space exploration missions. Medical ion therapy is another situation where human beings can be irradiated by heavy ions, usually to treat cancer. Heavy ions have a peculiar track structure characterized by high levels of energy ]deposition clustering, especially in near the track ends in the so ]called eBragg peak f region. In radiotherapy, these features of heavy ions can provide an improved dose conformation with respect to photons, also considering that the relative biological effectiveness (RBE) of therapeutic ions in the plateau region before the peak is sufficiently low. Therefore, several proton and carbon ion therapy facilities are under construction at this moment

Plante, Ianik↗

Demonstration of prominent actin filaments in the root columella

The distribution of actin filaments within the gravity-sensing columella cells of plant roots remains poorly understood, with studies over numerous years providing inconsistent descriptions of actin organization in these cells. This uncertainty in actin organization, and thus in actin's role in graviperception and gravisignaling, has led us to investigate actin arrangements in the columella cells of Zea mays L., Medicago truncatula Gaertn., Linum usitatissiilium L. and Nicotianla benthamiana Domin. Actin organization was examined using a combination of optimized immunofluorescence techniques, and an improved fluorochrome-conjugated phalloidin labeling method reliant on 3-maleimidobenzoyl-N-hydroxy-succinimide ester (MBS) cross-linking combined with glycerol permeabilization. Confocal microscopy of root sections labeled with anti-actin antibodies revealed patterns suggestive of actin throughout the columella region. These patterns included short and fragmented actin bundles, fluorescent rings around amyloplasts and intense fluorescence originating from the nucleus. Additionally, confocal microscopy of MBS-stabilized and Alexa Fluor-phalloidin-labeled root sections revealed a previously undetected state of actin organization in the columella. Discrete actin structures surrounded the amyloplasts and prominent actin cables radiated from the nuclear surface toward the cell periphery. Furthermore, the cortex of the columella cells contained fine actin bundles (or single filaments) that had a predominant transverse orientation. We also used confocal microscopy of plant roots expressing endoplasmic reticulum (ER)-targeted green fluorescent protein to demonstrate rapid ER movements within the columella cells, suggesting that the imaged actin network is functional. The successful identification of discrete actin structures in the root columella cells forms the perception and signaling.

Non-NASA Center↗

Astrobiology: The Search for Life in the Universe

Each of the 11 lead members of NASA's Astrobiology Institute has a specific mission. According to Hiroshi Ohmoto, director of Penn State s Astrobiology Research Center, Here we are mainly concerned with the origin of life and the evolution and extinction of important organisms. These include bacteria that live on methane, cyanobacteria (the inventors of photosynthesis), eukaryotes (a big category, covering anything with a nucleus, from single-celled organisms to humans), land-dwelling organisms, and early animals. Penn State astrobiologists are studying the environment before there was life on Earth, the origin of oxygen in the atmosphere, the chemical and thermal structures of oceans, and the role of metals in the evolution of life. Overall, they want to understand the connection between changes in environment and changes in life forms in the early Earth. PSARC offers research assistantships for graduate and undergraduate students, fellowships for graduate students and post-doctoral fellows, and an undergraduate minor in astrobiology. The minor covers 18 credits in earth sciences, geochemistry, geophysics, astronomy, biology, biochemistry, meteorology, and microbiology. The goal, says Ohmoto, is to teach students to critically evaluate claims related to this field that they encounter well after their college education has ended. Under a scanning electron microscope, Martian meteorite ALH84001 yields tube-like structures that look a lot like remnants of Earthly bacteria except smaller by a factor of ten.

Pacchioli, David↗

How cells (might) sense microgravity

This article is a summary of a lecture presented at an ESA/NASA Workshop on Cell and Molecular Biology Research in Space that convened in Leuven, Belgium, in June 1998. Recent studies are reviewed which suggest that cells may sense mechanical stresses, including those due to gravity, through changes in the balance of forces that are transmitted across transmembrane adhesion receptors that link the cytoskeleton to the extracellular matrix and to other cells (e.g., integrins, cadherins, selectins). The mechanism by which these mechanical signals are transduced and converted into a biochemical response appears to be based, in part, on the finding that living cells use a tension-dependent form of architecture, known as tensegrity, to organize and stabilize their cytoskeleton. Because of tensegrity, the cellular response to stress differs depending on the level of pre-stress (pre-existing tension) in the cytoskeleton and it involves all three cytoskeletal filament systems as well as nuclear scaffolds. Recent studies confirm that alterations in the cellular force balance can influence intracellular biochemistry within focal adhesion complexes that form at the site of integrin binding as well as gene expression in the nucleus. These results suggest that gravity sensation may not result from direct activation of any single gravioreceptor molecule. Instead, gravitational forces may be experienced by individual cells in the living organism as a result of stress-dependent changes in cell, tissue, or organ structure that, in turn, alter extracellular matrix mechanics, cell shape, cytoskeletal organization, or internal pre-stress in the cell-tissue matrix.--Ingber, D. How cells (might) sense microgravity.

NASA Discipline Cell Biology↗

Light-modulated abundance of an mRNA encoding a calmodulin-regulated, chromatin-associated NTPase in pea

A CDNA encoding a 47 kDa nucleoside triphosphatase (NTPase) that is associated with the chromatin of pea nuclei has been cloned and sequenced. The translated sequence of the cDNA includes several domains predicted by known biochemical properties of the enzyme, including five motifs characteristic of the ATP-binding domain of many proteins, several potential casein kinase II phosphorylation sites, a helix-turn-helix region characteristic of DNA-binding proteins, and a potential calmodulin-binding domain. The deduced primary structure also includes an N-terminal sequence that is a predicted signal peptide and an internal sequence that could serve as a bipartite-type nuclear localization signal. Both in situ immunocytochemistry of pea plumules and immunoblots of purified cell fractions indicate that most of the immunodetectable NTPase is within the nucleus, a compartment proteins typically reach through nuclear pores rather than through the endoplasmic reticulum pathway. The translated sequence has some similarity to that of human lamin C, but not high enough to account for the earlier observation that IgG against human lamin C binds to the NTPase in immunoblots. Northern blot analysis shows that the NTPase MRNA is strongly expressed in etiolated plumules, but only poorly or not at all in the leaf and stem tissues of light-grown plants. Accumulation of NTPase mRNA in etiolated seedlings is stimulated by brief treatments with both red and far-red light, as is characteristic of very low-fluence phytochrome responses. Southern blotting with pea genomic DNA indicates the NTPase is likely to be encoded by a single gene.

NASA Discipline Number 40-50↗

Cytoskeleton-amyloplast interactions in sweet clover

The distribution of organelles within columella cells of sweet clover was examined by transmission electron microscopy following growth under static or clinorotating conditions. A developmentally conditioned polarity was observed, with a proximal location of the nucleus and a distal accumulation of the endoplasmic reticulum. This polarity was insensitive to clinorotation. In contrast, clinorotation altered the location of amyloplasts. Application of cytoskeletal poisons (colchicine, cytochalasin D, taxol, and phalloidin), especially during clinorotation, had interesting effects on the maintenance of columella cell polarity, with a profound effect on the extent, location, and structure of the endoplasmic reticulum. The site of cytoskeletal interactions with sedimenting amyloplasts is thought to be the amyloplast envelope. An envelope fraction, having over 17 polypeptides, was isolated using immobilized antibody technology, and will provide a means of assessing the role of specific peptides in cytoskeleton/amyloplast interactions.

Non-NASA Center↗

Identification of amino acid sequences in the polyomavirus capsid proteins that serve as nuclear localization signals

The molecular mechanism participating in the transport of newly synthesized proteins from the cytoplasm to the nucleus in mammalian cells is poorly understood. Recently, the nuclear localization signal sequences (NLS) of many nuclear proteins have been identified, and most have been found to be composed of a highly basic amino acid stretch. A genetic "subtractive" and a biochemical "additive" approach were used in our studies to identify the NLS's of the polyomavirus structural capsid proteins. An NLS was identified at the N-terminus (Ala1-Pro-Lys-Arg-Lys-Ser-Gly-Val-Ser-Lys-Cys11) of the major capsid protein VP1 and at the C-terminus (Glu307 -Glu-Asp-Gly-Pro-Glu-Lys-Lys-Lys-Arg-Arg-Leu318) of the VP2/VP3 minor capsid proteins.

NASA Discipline Number 93-10↗

Interactions of Ku70/80 with Double-Strand DNA: Energetic, Dynamics, and Functional Implications

Space radiation is a proficient inducer of DNA damage leading to mutation, aberrant cell signaling, and cancer formation. Ku is among the first responding proteins in nucleus to recognize and bind the DNA double strand breaks (DSBs) whenever they are introduced. Once loaded Ku works as a scaffold to recruit other repair factors of non-homologous end joining and facilitates the following repair processes. The crystallographic study of the Ku70/80 heterodimer indicate the core structure of this protein shows virtually no conformational change after binding with DNA. To investigate the dynamical features as well as the energetic characteristics of Ku-DNA binding, we conduct multi-nanosecond molecular dynamics simulations of a modeled Ku70/80 structure and several complexes with two 24-bp DNA duplexes. Free energy calculations show significant energy differences between the complexes with Ku bound at DSBs and those with Ku associated at an internal site of a chromosome. The results also reveal detailed interactions between different nucleotides and the amino acids along the DNA-binding cradle of Ku, indicating subtle binding preference of Ku at specific DNA sequences. The covariance matrix analyses along the trajectories demonstrate the protein is stimulated to undergo correlated motions of different domains once bound to DNA ends. Additionally, principle component analyses identify these low frequency collective motions suitable for binding with and translocation along duplex DNA. It is proposed that the modification of dynamical properties of Ku upon binding with DSBs may provide a signal for the further recruitment of other repair factors such as DNA-PKcs, XLF, and XRCC4.

Hu, Shaowen↗

Simulation of the Formation of DNA Double Strand Breaks and Chromosome Aberrations in Irradiated Cells

The formation of DNA double-strand breaks (DSBs) and chromosome aberrations is an important consequence of ionizing radiation. To simulate DNA double-strand breaks and the formation of chromosome aberrations, we have recently merged the codes RITRACKS (Relativistic Ion Tracks) and NASARTI (NASA Radiation Track Image). The program RITRACKS is a stochastic code developed to simulate detailed event-by-event radiation track structure: [1] This code is used to calculate the dose in voxels of 20 nm, in a volume containing simulated chromosomes, [2] The number of tracks in the volume is calculated for each simulation by sampling a Poisson distribution, with the distribution parameter obtained from the irradiation dose, ion type and energy. The program NASARTI generates the chromosomes present in a cell nucleus by random walks of 20 nm, corresponding to the size of the dose voxels, [3] The generated chromosomes are located within domains which may intertwine, and [4] Each segment of the random walks corresponds to approx. 2,000 DNA base pairs. NASARTI uses pre-calculated dose at each voxel to calculate the probability of DNA damage at each random walk segment. Using the location of double-strand breaks, possible rejoining between damaged segments is evaluated. This yields various types of chromosomes aberrations, including deletions, inversions, exchanges, etc. By performing the calculations using various types of radiations, it will be possible to obtain relative biological effectiveness (RBE) values for several types of chromosome aberrations.

Plante, Ianik↗

Monte Carlo mixture model of lifetime cancer incidence risk from radiation exposure on shuttle and international space station

Estimating uncertainty in lifetime cancer risk for human exposure to space radiation is a unique challenge. Conventional risk assessment with low-linear-energy-transfer (LET)-based risk from Japanese atomic bomb survivor studies may be inappropriate for relativistic protons and nuclei in space due to track structure effects. This paper develops a Monte Carlo mixture model (MCMM) for transferring additive, National Institutes of Health multiplicative, and multiplicative excess cancer incidence risks based on Japanese atomic bomb survivor data to determine excess incidence risk for various US astronaut exposure profiles. The MCMM serves as an anchor point for future risk projection methods involving biophysical models of DNA damage from space radiation. Lifetime incidence risks of radiation-induced cancer for the MCMM based on low-LET Japanese data for nonleukemia (all cancers except leukemia) were 2.77 (90% confidence limit, 0.75-11.34) for males exposed to 1 Sv at age 45 and 2.20 (90% confidence limit, 0.59-10.12) for males exposed at age 55. For females, mixture model risks for nonleukemia exposed separately to 1 Sv at ages of 45 and 55 were 2.98 (90% confidence limit, 0.90-11.70) and 2.44 (90% confidence limit, 0.70-10.30), respectively. Risks for high-LET 200 MeV protons (LET=0.45 keV/micrometer), 1 MeV alpha-particles (LET=100 keV/micrometer), and 600 MeV iron particles (LET=180 keV/micrometer) were scored on a per particle basis by determining the particle fluence required for an average of one particle per cell nucleus of area 100 micrometer(2). Lifetime risk per proton was 2.68x10(-2)% (90% confidence limit, 0.79x10(-3)%-0. 514x10(-2)%). For alpha-particles, lifetime risk was 14.2% (90% confidence limit, 2.5%-31.2%). Conversely, lifetime risk per iron particle was 23.7% (90% confidence limit, 4.5%-53.0%). Uncertainty in the DDREF for high-LET particles may be less than that for low-LET radiation because typically there is very little dose-rate dependence. Probability density functions for high-LET radiation quality and dose-rate may be preferable to conventional risk assessment approaches. Nuclear reactions and track structure effects in tissue may not be properly estimated by existing data using in vitro models for estimating RBEs. The method used here is being extended to estimate uncertainty in spacecraft shielding effectiveness in various space radiation environments.

NASA Center LaRC↗

Radioprotection of Human Cell Nuclear DNA by Polyamines: Radiosensitivity of Chromatin is Influenced by Tightly Bound Spermine

The polyamines putrescine (PUT) and spermine (SPM) were examined for their ability to protect human cell Deoxyribonucleic Acid (DNA) against the formation of radiation-induced double-strand breaks (DSBs). As observed previously, under conditions where polyamines were shown to be almost completely absent, association with nuclear matrix protein into a nucleoid, and organization into chromatin structure, protected DNA from induction of DSBs by factors of 4.5 and 95, respectively. At concentrations below 1 mM, PUT or SPM provided equivalent levels of protection to deproteinized nuclear DNA, consistent with their capacity to scavenge radiation-induced radicals. At constant ionic strength, 5 mM SPM protected deproteinized DNA and nucleoid DNA and DNA in nuclear chromatin by factors of 100 and 26, respectively. At 5 mM, SPM provided 15 times greater protection of deproteinized DNA than did PUT. Under physiologically relevant conditions, 5 mM SPM protected DNA in the intact nucleus from the induction of DSBs by a factor of 2 relative to DNA in the absence of SPM. Studies of SPM binding during cellular fractionation revealed that a significant fraction of the cellular SPM is tightly bound in the nucleus but can be removed by extended washing. Thus the association of SPM with nuclear chromatin appears to be a significant contributor to the resistance of the cell's DNA to the induction of DSBs.

Warters, Raymond L.↗

The origin of the eukaryotic cell

The endosymbiotic hypothesis for the origin of the eukaryotic cell has been applied to the origin of the mitochondria and chloroplasts. However as has been pointed out by Mereschowsky in 1905, it should also be applied to the nucleus as well. If the nucleus, mitochondria and chloroplasts are endosymbionts, then it is likely that the organism that did the engulfing was not a DNA-based organism. In fact, it is useful to postulate that this organism was a primitive RNA-based organism. This hypothesis would explain the preponderance of RNA viruses found in eukaryotic cells. The centriole and basal body do not have a double membrane or DNA. Like all MTOCs (microtubule organising centres), they have a structural or morphic RNA implicated in their formation. This would argue for their origin in the early RNA-based organism rather than in an endosymbiotic event involving bacteria. Finally, the eukaryotic cell uses RNA in ways quite unlike bacteria, thus pointing to a greater emphasis of RNA in both control and structure in the cell. The origin of the eukaryotic cell may tell us why it rather than its prokaryotic relative evolved into the metazoans who are reading this paper.

Review↗

Fine structural changes in the lateral vestibular nucleus of aging rats

The fine structure of the lateral vestibular nucleus was investigated in Sprague-Dawley rats, that were sacrified at 4 weeks, 6-8 weeks, 6-8 months, and 18-20 months of age. In the neuronal perikaria, the following age-associated changes were seen with increasing frequency with advancing age: rodlike nuclear inclusions and nuclear membrane invaginations; cytoplasmic dense bodies with the characteristics of lipofuscin; and moderate disorganization of the granular endoplasmic reticulum. Dense bodies were also seen in glial cells. Rats 18 to 20 months old showed dendritic swellings, axonal degeneration, and an apparent increase in the number of axosomatic synaptic terminals containing flattened vesicles (presumed to be inhibitory in function).

Johnson, J. E., Jr.↗

Cortical and cap sedimentation in gravitropic Equisetum roots

Although the rootcap is required for gravitropic sensing, various classical and contemporary data raise the question of whether additional sensing occurs away from the cap in roots. Roots of Equisetum hyemale L. (horsetail) were examined by light and electron microscopy to determine which cell components were distributed with respect to gravity both in and away from the rootcap. Adventitious roots from stem cuttings were gravitropic in a vertical orientation or if reoriented to the horizontal. Obvious amyloplast sedimentation was found in vertical and in reoriented roots 1) in cells in the center of the rootcap and 2) in young, elongating cortical cells located in two to three layers outside the endodermis. These cortical amyloplasts were smaller than cap amyloplasts and, unlike central cap amyloplasts, were occasionally found in the top of the cell. The nucleus was also sedimented on top of the amyloplasts in both cell types, both in vertical and in reoriented roots. Sedimentation of both organelles ceased as cortical cells elongated further or as cap cells became peripheral in location. In both cell types with sedimentation, endoplasmic reticulum was located in the cell periphery, but showed no obvious enrichment near the lower part of the cell in vertical roots. This is the first modern report of sedimentation away from the cap in roots, and it provides structural evidence that gravitropic sensing may not be confined to the cap in all roots.

NASA Discipline Plant Biology↗

The Analysis of the Patterns of Radiation-Induced DNA Damage Foci by a Stochastic Monte Carlo Model of DNA Double Strand Breaks Induction by Heavy Ions and Image Segmentation Software

To create a generalized mechanistic model of DNA damage in human cells that will generate analytical and image data corresponding to experimentally observed DNA damage foci and will help to improve the experimental foci yields by simulating spatial foci patterns and resolving problems with quantitative image analysis. Material and Methods: The analysis of patterns of RIFs (radiation-induced foci) produced by low- and high-LET (linear energy transfer) radiation was conducted by using a Monte Carlo model that combines the heavy ion track structure with characteristics of the human genome on the level of chromosomes. The foci patterns were also simulated in the maximum projection plane for flat nuclei. Some data analysis was done with the help of image segmentation software that identifies individual classes of RIFs and colocolized RIFs, which is of importance to some experimental assays that assign DNA damage a dual phosphorescent signal. Results: The model predicts the spatial and genomic distributions of DNA DSBs (double strand breaks) and associated RIFs in a human cell nucleus for a particular dose of either low- or high-LET radiation. We used the model to do analyses for different irradiation scenarios. In the beam-parallel-to-the-disk-of-a-flattened-nucleus scenario we found that the foci appeared to be merged due to their high density, while, in the perpendicular-beam scenario, the foci appeared as one bright spot per hit. The statistics and spatial distribution of regions of densely arranged foci, termed DNA foci chains, were predicted numerically using this model. Another analysis was done to evaluate the number of ion hits per nucleus, which were visible from streaks of closely located foci. In another analysis, our image segmentaiton software determined foci yields directly from images with single-class or colocolized foci. Conclusions: We showed that DSB clustering needs to be taken into account to determine the true DNA damage foci yield, which helps to determine the DSB yield. Using the model analysis, a researcher can refine the DSB yield per nucleus per particle. We showed that purely geometric artifacts, present in the experimental images, can be analytically resolved with the model, and that the quantization of track hits and DSB yields can be provided to the experimentalists who use enumeration of radiation-induced foci in immunofluorescence experiments using proteins that detect DNA damage. An automated image segmentaiton software can prove useful in a faster and more precise object counting for colocolized foci images.

Ponomarev, Artem↗

Tracks to therapy

Studies of the structure of particle tracks have led to models of track effects based on radial dose and radiobiological target theory that have been very successful in describing and predicting track effects in physical, chemical, and biological systems. For describing mammalian cellular inactivation two inactivation modes are required, called gamma-kill and ion-kill, the first due to synergistic effects of delta rays from adjacent ion paths thus resembling the effects from gamma rays, and the second to the effects of single ion transits through a cell nucleus. The ion-kill effect is more severe, where the fraction of cells experiencing ion kill is responsible for a decrease in the oxygen enhancement ratio, and an increase in relative biological effectiveness, but these are accompanied by loss of repair, hence to a reduction in the efficiency of fractionation in high LET therapy, as shown by our calculations for radiobiological effects in the "spread out Bragg Peak".

Review, Tutorial↗

Expression and purification of recombinant polyomavirus VP2 protein and its interactions with polyomavirus proteins

Recombinant polyomavirus VP2 protein was expressed in Escherichia coli (RK1448), using the recombinant expression system pFPYV2. Recombinant VP2 was purified to near homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electroelution, and Extracti-Gel chromatography. Polyclonal serum to this protein which reacted specifically with recombinant VP2 as well as polyomavirus virion VP2 and VP3 on Western blots (immunoblots) was produced. Purified VP2 was used to establish an in vitro protein-protein interaction assay with polyomavirus structural proteins and purified recombinant VP1. Recombinant VP2 interacted with recombinant VP1, virion VP1, and the four virion histones. Recombinant VP1 coimmunoprecipitated with recombinant VP2 or truncated VP2 (delta C12VP2), which lacked the carboxy-terminal 12 amino acids. These experiments confirmed the interaction between VP1 and VP2 and revealed that the carboxyterminal 12 amino acids of VP2 and VP3 were not necessary for formation of this interaction. In vivo VP1-VP2 interaction study accomplished by cotransfection of COS-7 cells with VP2 and truncated VP1 (delta N11VP1) lacking the nuclear localization signal demonstrated that VP2 was capable of translocating delta N11VP1 into the nucleus. These studies suggest that complexes of VP1 and VP2 may be formed in the cytoplasm and cotransported to the nucleus for virion assembly to occur.

Non-NASA Center↗

Modeling the Activity of Single Genes

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.

Mjolsness, Eric↗