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At least 307 records · Page 17

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NASA NTRS2000

Caveolin-1 regulates shear stress-dependent activation of extracellular signal-regulated kinase

Fluid shear stress activates a member of the mitogen-activated protein (MAP) kinase family, extracellular signal-regulated kinase (ERK), by mechanisms dependent on cholesterol in the plasma membrane in bovine aortic endothelial cells (BAEC). Caveolae are microdomains of the plasma membrane that are enriched with cholesterol, caveolin, and signaling molecules. We hypothesized that caveolin-1 regulates shear activation of ERK. Because caveolin-1 is not exposed to the outside, cells were minimally permeabilized by Triton X-100 (0.01%) to deliver a neutralizing, polyclonal caveolin-1 antibody (pCav-1) inside the cells. pCav-1 then bound to caveolin-1 and inhibited shear activation of ERK but not c-Jun NH(2)-terminal kinase. Epitope mapping studies showed that pCav-1 binds to caveolin-1 at two regions (residues 1-21 and 61-101). When the recombinant proteins containing the epitopes fused to glutathione-S-transferase (GST-Cav(1-21) or GST-Cav(61-101)) were preincubated with pCav-1, only GST-Cav(61-101) reversed the inhibitory effect of the antibody on shear activation of ERK. Other antibodies, including m2234, which binds to caveolin-1 residues 1-21, had no effect on shear activation of ERK. Caveolin-1 residues 61-101 contain the scaffolding and oligomerization domains, suggesting that binding of pCav-1 to these regions likely disrupts the clustering of caveolin-1 or its interaction with signaling molecules involved in the shear-sensitive ERK pathway. We suggest that caveolae-like domains play a critical role in the mechanosensing and/or mechanosignal transduction of the ERK pathway.

Non-NASA Center↗

NASA NTRS1999

Cross-linking staphylococcal enterotoxin A bound to major histocompatibility complex class I is required for TNF-alpha secretion

The mechanism of how superantigens function to activate cells has been linked to their ability to bind and cross-link the major histocompatibility complex class II (MHCII) molecule. Cells that lack the MHCII molecule also respond to superantigens, however, with much less efficiency. Therefore, the purpose of this study was to confirm that staphylococcal enterotoxin A (SEA) could bind the MHCI molecule and to test the hypothesis that cross-linking SEA bound to MHCII-deficient macrophages would induce a more robust cytokine response than without cross-linking. We used a capture enzyme-linked immunosorbent assay and an immunprecipitation assay to directly demonstrate that MHCI molecules bind SEA. Directly cross-linking MHCI using monoclonal antibodies or cross-linking bound SEA with an anti-SEA antibody or biotinylated SEA with avidin increased TNF-alpha and IL-6 secretion by MHCII(-/-) macrophages. The induction of a vigorous macrophage cytokine response by SEA/anti-SEA cross-linking of MHCI offers a mechanism to explain how MHCI could play an important role in superantigen-mediated pathogenesis. Copyright 1999 Academic Press.

NASA Discipline Musculoskeletal↗

NASA NTRS1999

Cloning the promoter for transforming growth factor-beta type III receptor. Basal and conditional expression in fetal rat osteoblasts

Transforming growth factor-beta binds to three high affinity cell surface molecules that directly or indirectly regulate its biological effects. The type III receptor (TRIII) is a proteoglycan that lacks significant intracellular signaling or enzymatic motifs but may facilitate transforming growth factor-beta binding to other receptors, stabilize multimeric receptor complexes, or segregate growth factor from activating receptors. Because various agents or events that regulate osteoblast function rapidly modulate TRIII expression, we cloned the 5' region of the rat TRIII gene to assess possible control elements. DNA fragments from this region directed high reporter gene expression in osteoblasts. Sequencing showed no consensus TATA or CCAAT boxes, whereas several nuclear factors binding sequences within the 3' region of the promoter co-mapped with multiple transcription initiation sites, DNase I footprints, gel mobility shift analysis, or loss of activity by deletion or mutation. An upstream enhancer was evident 5' proximal to nucleotide -979, and a silencer region occurred between nucleotides -2014 and -2194. Glucocorticoid sensitivity mapped between nucleotides -687 and -253, whereas bone morphogenetic protein 2 sensitivity co-mapped within the silencer region. Thus, the TRIII promoter contains cooperative basal elements and dispersed growth factor- and hormone-sensitive regulatory regions that can control TRIII expression by osteoblasts.

Non-NASA Center↗

NASA NTRS1992

Availability of the B beta(15-21) epitope on cross-linked human fibrin and its plasmic degradation products

The binding of radiolabeled monoclonal antifibrin antibody 59D8 (specific for fibrin but not fibrinogen) to a series of degraded fibrin clots showed that the availability of the B beta(15-21) epitope (against which 59D8 had been raised) was inversely proportional to the extent of clot lysis. Examination of digest supernatants revealed that the B beta(15-21) epitope was released from clots as a high molecular weight degradation product in the presence of calcium ions but that the generation of low molecular weight peptides occurred in the absence of calcium ions. To address the question of epitope accessibility, we compared levels of fibrin clot binding among four radioactively labeled antibodies: antifibrin monoclonal antibody 59D8, two antifibrinogen monoclonal antibodies that cross-reacted with fibrin, and an affinity-purified polyclonal antifibrinogen antibody. We expected that the antifibrinogen antibodies would show enhanced binding to clots in comparison with the antifibrin antibody. However, the epitope accessibility experiments showed that all four antibody preparations bound fibrin clots at comparable levels. Taken together, these studies demonstrated that one fibrin-specific epitope, B beta(15-21), remains available on clots as they undergo degradation by plasmin and, importantly, that the epitope is not solubilized at a rate faster than the rate at which the clot is itself solubilized. The availability of the B beta(15-21) epitope during the course of plasminolysis assures the potential utility of antifibrin antibodies such as 59D8 for detecting thrombi and targeting plasminogen activators.

NASA Discipline Regulatory Physiology↗

NASA NTRS2005

Conservation of Fold and Topology of Functional Elements in Thiamin Pyrophosphate Enzymes

Thiamin pyrophosphate (TPP)-dependent enzymes are a highly divergent family of proteins binding both TPP and metal ions. They perform decarboxylation-hydroxyaldehydes. Prior -ketoacids and of a common - (O=)C-C(OH)- fragment of to knowledge of three-dimensional structures of these enzmes, the GDGY25-30NN sequence was used to identify these enzymes. Subsequently, a number of structural studies on those enzymes revealed multi-subunit organization and the features of the two duplicate cofactor binding sites. Analyzing the structures of 44 structurally known enzymes, we found that the common structure of these enzymes is reduced to 180-220 amino acid long fragments of two PP and two PYR domains that form the [PP:PYR]2 binding center of two cofactor molecules. The structures of PP and PYR are arranged in a similar fold-sheet with triplets of helices on both sides.Dconsisting of a six-stranded Residues surrounding the cofactors are not strictly conserved, but they provide the same interatomic contacts required for the catalytic functions that these enzymes perform while maintaining interactive structural integrity. These structural and functional amino acids are topological counterparts located in the same positions of the conserved fold of sets of PP and PYR domains. Additional parallels include short fragments of sequences that link these amino acids to the fold and function. This report on the structural commonalities amongst TPP dependent enzymes is thought to contribute new approaches to annotation that may assist in advancing the functional proteomics of TPP dependent enzymes, and trace their complexity within evolutionary context.

Dominiak, P.↗

NASA NTRS2004

Computational studies of Ras and PI3K

Until recently, experimental techniques in molecular cell biology have been the primary means to investigate biological risk upon space radiation. However, computational modeling provides an alternative theoretical approach, which utilizes various computational tools to simulate proteins, nucleotides, and their interactions. In this study, we are focused on using molecular mechanics (MM) and molecular dynamics (MD) to study the mechanism of protein-protein binding and to estimate the binding free energy between proteins. Ras is a key element in a variety of cell processes, and its activation of phosphoinositide 3-kinase (PI3K) is important for survival of transformed cells. Different computational approaches for this particular study are presented to calculate the solvation energies and binding free energies of H-Ras and PI3K. The goal of this study is to establish computational methods to investigate the roles of different proteins played in the cellular responses to space radiation, including modification of protein function through gene mutation, and to support the studies in molecular cell biology and theoretical kinetics models for our risk assessment project.

Ren, Lei↗

NASA NTRS2009

A Model for Predicting Thermoelectric Properties of Bi2Te3

A parameterized orthogonal tight-binding mathematical model of the quantum electronic structure of the bismuth telluride molecule has been devised for use in conjunction with a semiclassical transport model in predicting the thermoelectric properties of doped bismuth telluride. This model is expected to be useful in designing and analyzing Bi2Te3 thermoelectric devices, including ones that contain such nano - structures as quantum wells and wires. In addition, the understanding gained in the use of this model can be expected to lead to the development of better models that could be useful for developing other thermoelectric materials and devices having enhanced thermoelectric properties. Bi2Te3 is one of the best bulk thermoelectric materials and is widely used in commercial thermoelectric devices. Most prior theoretical studies of the thermoelectric properties of Bi2Te3 have involved either continuum models or ab-initio models. Continuum models are computationally very efficient, but do not account for atomic-level effects. Ab-initio models are atomistic by definition, but do not scale well in that computation times increase excessively with increasing numbers of atoms. The present tight-binding model bridges the gap between the well-scalable but non-atomistic continuum models and the atomistic but poorly scalable ab-initio models: The present tight-binding model is atomistic, yet also computationally efficient because of the reduced (relative to an ab-initio model) number of basis orbitals and flexible parameterization of the Hamiltonian.

Lee, Seungwon↗

NASA NTRS2007

Antibodies Against Three Forms of Urokinase

Antibodies that bind to preselected regions of the urokinase molecule have been developed. These antibodies can be used to measure small quantities of each of three molecular forms of urokinase that could be contained in microsamples or conditioned media harvested from cultures of mammalian cells. Previously available antibodies and assay techniques do not yield both clear distinctions among, and measurements of, all three forms. Urokinase is a zymogen that is synthesized in a single-chain form, called ScuPA, which is composed of 411 amino acid residues (see figure). ScuPA has very little enzyme activity, but it can be activated in two ways: (1) by cleavage of the peptide bond lysine 158/isoleucine 159 and the loss of lysine 158 to obtain the high molecular-weight (HMW) form of the enzyme or (2) by cleavage of the bond lysine 135/lysine 136 to obtain the low-molecular-weight (LMW) form of the enzyme. The antibodies in question were produced in mice and rabbits by use of peptides as immunogens. The peptides were selected to obtain antibodies that bind to regions of ScuPA that include the lysine 158/isoleucine 159 and the lysine 135/lysine 136 bonds. The antibodies include monoclonal and polyclonal ones that yield indications as to whether either of these bonds is intact. The polyclonal antibodies include ones that preferentially bind to the HMW or LMW forms of the urokinase molecule. The monoclonal antibodies include ones that discriminate between the ScuPA and the HMW form. A combination of these molecular-specific antibodies will enable simultaneous assays of the ScuPA, HMW, and LMW forms in the same specimen of culture medium.

Morrison, Dennis R.↗

NASA NTRS2011

Atomic Simulation of Complex DNA DSBs and the Interactions with the Ku70/80 Heterodimer

DNA double strand breaks (DSBs) induced by ionizing radiation (IR) usually contain modified bases such as 8-oxo-7,8-dihydroguanine (8-oxoG) and thymine glycol, apurinic/apyrimidinic (AP) sites, 2-deoxyribonolactone, or single-strand breaks (SSBs). The presence of such lesions in close proximity to the DSB terminus makes the DNA nicks more difficult to repair and rejoin than endogenously induced simple DSBs, and as such a major determinant of the biological effects of high linear energy transfer (LET) radiation as encountered in space travel. In this study we conducted molecular dynamics simulations on a series of DNA duplexes with various complex lesions of 8-oxoG and AP sites, in an effort to investigate the effects of such lesions to the structural integrity and stability of DNA after insulted by IR. We also simulated the interaction of such complex DSBs with the Ku70/80 heterodimer, the first protein in mammalian cells to embark the non-homologous end joining (NHEJ) DNA repair pathway. The results indicate, compared to DNA with simple DSBs, the complex lesions can enhance the hydrogen bonds opening rate at the DNA terminus, and increase the mobility of the whole duplex, thus they present more deleterious effects to the genome integrity if not captured and repaired promptly in cells. Simulations also demonstrate the binding of Ku drastically reduces structural disruption and flexibility caused by the complex lesions, and the interactions of Ku with complex DSBs have a different potential energy landscape from the bound structure with simple DSB. In all complex DSBs systems, the binding of DSB terminus with Ku70 is softened while the binding of the middle duplex with Ku80 is tightened. This energy shift may help the Ku protein to secure at the DSB terminus for a longer time, so that other end processing factors or repair pathways can proceed at the lesions before NHEJ repair process starts. These atomic simulations may provide valuable new insight into the selective action of repair proteins on damaged DNA.

Hu, Shaowen↗

NASA NTRS2012

Molecular Technique to Reduce PCR Bias for Deeper Understanding of Microbial Diversity

Current planetary protection policies require that spacecraft targeted to sensitive solar system bodies be assembled and readied for launch in controlled cleanroom environments. A better understanding of the distribution and frequency at which high-risk contaminant microbes are encountered on spacecraft surfaces would significantly aid in assessing the threat of forward contamination. However, despite a growing understanding of the diverse microbial populations present in cleanrooms, less abundant microbial populations are probably not adequately taken into account due to technological limitations. This novel approach encompasses a wide spectrum of microbial species and will represent the true picture of spacecraft cleanroom-associated microbial diversity. All of the current microbial diversity assessment techniques are based on an initial PCR amplification step. However, a number of factors are known to bias PCR amplification and jeopardize the true representation of bacterial diversity. PCR amplification of a minor template appears to be suppressed by the amplification of a more abundant template. It is widely acknowledged among environmental molecular microbiologists that genetic biosignatures identified from an environment only represent the most dominant populations. The technological bottleneck overlooks the presence of the less abundant minority population and may underestimate their role in the ecosystem maintenance. DNA intercalating agents such as propidium monoazide (PMA) covalently bind with DNA molecules upon photolysis using visible light, and make it unavailable for DNA polymerase enzyme during polymerase chain reaction (PCR). Environmental DNA samples will be treated with suboptimum PMA concentration, enough to intercalate with 90 99% of the total DNA. The probability of PMA binding with DNA from abundant bacterial species will be much higher than binding with DNA from less abundant species. This will increase the relative DNA concentration of previously "shadowed" less abundant species available for PCR amplification. These PCR products obtained with and without PMA treatment will then be subjected to downstream diversity analyses such as sequencing and DNA microarray. It is expected that PMA-coupled PCR will amplify the "minority population" and help in understanding microbial diversity spectrum of an environmental sample at a much deeper level. This new protocol aims to overcome the major potential biases faced when analyzing microbial 16S rRNA gene diversity. This study will lead to a technological advancement and a commercial product that will aid microbial ecologists in understanding microbial diversity from various environmental niches. Implementation of this technique may lead to discoveries of novel microbes and their functions in sustenance of the ecosystem.

Vaishampayan, Parag A.↗

NASA NTRS2013

FRET-Aptamer Assays for Bone Marker Assessment, C-Telopeptide, Creatinine, and Vitamin D

Astronauts lose 1.0 to 1.5% of their bone mass per month on long-duration spaceflights. NASA wishes to monitor the bone loss onboard spacecraft to develop nutritional and exercise countermeasures, and make adjustments during long space missions. On Earth, the same technology could be used to monitor osteoporosis and its therapy. Aptamers bind to targets against which they are developed, much like antibodies. However, aptamers do not require animal hosts or cell culture and are therefore easier, faster, and less expensive to produce. In addition, aptamers sometimes exhibit greater affinity and specificity vs. comparable antibodies. In this work, fluorescent dyes and quenchers were added to the aptamers to enable pushbutton, one-step, bind-and-detect fluorescence resonance energy transfer (FRET) assays or tests that can be freeze-dried, rehydrated with body fluids, and used to quantitate bone loss of vitamin D levels with a handheld fluorometer in the spacecraft environment. This work generated specific, rapid, one-step FRET assays for the bone loss marker C-telopeptide (CTx) when extracted from urine, creatinine from urine, and vitamin D congeners in diluted serum. The assays were quantified in nanograms/mL using a handheld fluorometer connected to a laptop computer to convert the raw fluorescence values into concentrations of each analyte according to linear standard curves. DNA aptamers were selected and amplified for several rounds against a 26- amino acid form of CTx, creatinine, and vitamin D. The commonalities between loop structures were studied, and several common loop structures were converted into aptamer beacons with a fluorophore and quencher on each end. In theory, when the aptamer beacon binds its cognate target (CTx bone peptide, creatinine, or vitamin D), it is forced open and no longer quenched, so it gives off fluorescent light (when excited) in proportion to the amount of target present in a sample. This proportional increase in fluorescence is called a "lights on" FRET response. The vitamin D aptamer beacon gives a "lights off" or inversely proportional fluorescence response to the amount of vitamin D present in diluted serum. These FRET-aptamer assays are rapid (<30 minutes), sensitive (low ng/mL detection limits), and quite easy to carry out (add sample, mix, and detect in the handheld reader). Benefits include the speed of the assays as well as the small amount of space taken up by the handheld reader and cuvette assays. The aptamer DNA sequences represent novel additional features of the existing (patent-pending) FRET-aptamer assay platform.

Bruno, John G.↗

NASA NTRS2015

Biomaterials Out of Thin Air: in Situ, On-Demand Printing of Advanced Biocomposites

Upmass is the single most significant limitation of our current space mission capability. Although biomaterials and biocomposites have mass, strength, flexibility, and self-healing properties that could significantly reduce upmass, their use is limited by the following drawbacks: Expensive, specific production. Many biomaterials can only be produced as part of significant support ecosystem; Inaccessible functional customization. The grain of wood, the porosity of bone, and so on are an integral part of the materials' desired mechanical properties, but are not deterministic when the material is naturally grown; Limited compositions. Most biomaterials (unlike metal, plastic, etc.) cannot be easily combined or modified to produce new materials. This project builds on recent advances in: Synthetic biology. Libraries of standardized genetic parts which can be used for controlled cellular material production, delivery, and binding; 3D printing. Commercial off-the-shelf components which can be used to make of a pico- to nanoliter cell deposition system; Tissue engineering. Proven cell-compatible support hydrogels and scaffolds can be modified to bind the deposited biomaterials of interest. Objectives: Feasibility and benefit analysis. Two mission contexts span the concept's scope (see below); Proof-of-concept demonstration. A simple grid of two proteins, fluorescent for easy detection, to validate the core technology concept; Proposed implementations for follow-on work. Avenues for future work on each core component (host cell, production control, material delivery, material binding, etc.); Complementary studies exploration. A survey of other emerging areas (in situ resource utilization, protein engineering, etc.) with the potential to multiply our technology's impact. Potential Impacts: This application could dramatically expand manufacturing capabilities on Earth and in space: In situ resource utilization. A far greater range of materials and products will be available from the limited palette offered by in situ resource extraction techniques; Reduced equipment and material upmass for off-Earth habitats. Ready- to-use highly specialized construction materials (radiation hardened, compressive/tensile, light or dense) from an extremely low starting mass; Structured biomaterial production. New ready-to-use macro, micro, and molecular manufacturing techniques for traditional biomaterials such as wood, bone and shell; New and novel biocomposite creation. The ability to create completely novel material composites from any base material that cells can be engineered to produce. Suggested Mission Contexts: ISS part manufacturing. A 'minimal working example' making a finished biomaterial part aboard the International Space Station; A long-term Mars habitat. 'Cradle-to-grave' use at a hypothetical Mars habitat, covering everything from tools to construction materials. Alternate Abstract: Imagine being able to print anything from tools and composite building materials to food and human tissues. Imagine being on Mars with the ability to replace any broken part, whether it's a part of your spacesuit, your habitat, or your own body. We propose a technique that would allow just that. By printing 3D arrays of cells engineered to secrete the necessary materials, the abundant in situ resources of atmosphere and regolith become organic, inorganic, or organic-inorganic composite materials. Such materials include novel, biologically derived materials not previously possible to fabricate.

habitats↗

NASA NTRS2015

Biomaterials Out of Thin Air: in Situ, On-demand Printing of Advanced Biocomposites

habitats↗

NASA NTRSDate not supplied

Space Suit Portable Life Support System Fastener and Insert Summary, Issues, and Testing Results

Fasteners are included in every assembly on the Exploration External Mobility Unity Portable Life Support System (xEMU PLSS). However, fasteners and inserts have been the cause of many issues seen on the PLSS during development; furthermore, inserts with thermoplastics are not very well understood. The materials used in the PLSS are sometimes softer and lighter weight metals or thermoplastics which require the installation of inserts to improve the interface strength. In addition, space flight requirements call out the need for an additional running/locking torque, so they do not detach in case of failure of the final torque. The xEMU PLSS has experienced various issues with fasteners and inserts, from the bolt binding in an insert or the running torque failing prematurely. A study has been completed to address the running torque life problem. This study compares the insert and locking feature combinations to determine the combination which maximizes life. The study found that using a locking patch with an insert fails after the first installation. Therefore, it is no longer recommended to use a locking patch fastener with an insert for future installments. For the bolt binding issue, inserts have been installed too deep and have caused the bolt to bind near the final threads of the inserts where the hole is not tapped deep enough. Thermoplastics, on the other end, can either use standard helical inserts or heat set inserts. Another study was performed using tangless inserts and heat inserts installed in thermoplastics to quantify the expected pull-out strength for various insert installation parameters (insert type, temperature, and pressure). This paper will review the issues and lessons learned with fasteners on the xEMU PLSS, provide results of a locking feature study, and provide results and recommended installation settings from a heat insert study.

Ryan Ogilvie↗

NASA NTRS2021

Frustration in Fuzzy Protein Complexes Leads to Interaction Versatility

Disordered proteins frequently serve as interaction hubs involving a constrained variety of partners. Complexes with different partners frequently exhibit distinct binding modes, involving regions that remain disordered in the bound state. While the conformational properties of disordered proteins are well-characterized in their free states, less is known about the molecular mechanisms by which specificity can be achieved not with one but with multiple partners. Using the energy landscape theory concept of protein frustration, we demonstrate that complexes of disordered proteins exhibit a high degree of local frustration, especially at the binding interface. These suboptimal interactions lead to the possibility of multiple bound substates, each displaying distinct frustration patterns, which are differently populated in complexes with different partners. These results explain how specificity of disordered proteins can be achieved without a single common bound conformation and how the conflict between different interactions can be used to control the binding to multiple partners.

Chemical specificity↗

NASA NTRS2022

Overcoming Universal Restrictions on Metal Selectivity by Protein Design

Selective metal coordination is central to the functions of metalloproteins:1,2 each metalloprotein must pair with its cognate metallocofactor to fulfl its biological role3 . However, achieving metal selectivity solely through a three-dimensional protein structure is a great challenge, because there is a limited set of metal-coordinating amino acid functionalities and proteins are inherently fexible, which impedes steric selection of metals3,4 . Metal-binding afnities of natural proteins are primarily dictated by the electronic properties of metal ions and follow the Irving–Williams series5 (Mn2+ < Fe2+ < Co2+ < Ni2+ Zn2+) with few exceptions6,7 . Accordingly, metalloproteins overwhelmingly bind Cu2+ and Zn2+ in isolation, regardless of the nature of their active sites and their cognate metal ions1,3,8 . This led organisms to evolve complex homeostatic machinery and non-equilibrium strategies to achieve correct metal speciation1,3,8–10. Here we report an artifcial dimeric protein, (AB)2, that thermodynamically overcomes the Irving–Williams restrictions in vitro and in cells, favouring the binding of lower-Irving–Williams transition metals over Cu2+, the most dominant ion in the Irving–Williams series. Counter to the convention in molecular design of achieving specifcity through structural preorganization, (AB)2 was deliberately designed to be fexible. This fexibility enabled (AB)2 to adopt mutually exclusive, metal-dependent conformational states, which led to the discovery of structurally coupled coordination sites that disfavour Cu2+ ions by enforcing an unfavourable coordination geometry. Aside from highlighting fexibility as a valuable element in protein design, our results illustrate design principles for constructing selective metal sequestration agents.

Tae Su Choi↗

NASA NTRS1992

Crosslinking transcription factors to their recognition sequences with PtII complexes

We have prepared phosphorothioate-containing cyclic oligodeoxynucleotides that fold into 'dumbbells' containing CRE and TRE sequences, the binding sequences for the CREB and JUN proteins, respectively. Six phosphorothioate residues were introduced into each of the recognition sequences. K2PtCl4 crosslinks CRE to CREB and TRE to JUN. The extent of crosslinking is about eight times greater than that observed with standard oligodeoxynucleotides and amounts to 30-50% of the efficiency of non-covalent association as estimated by gel-shift assays. Crosslinking is reversed by incubation with NaCN. The crosslinking reaction is specific--a dumbbell oligonucleotide with six phosphorothioate groups introduced into the Sp1 recognition sequence could not be crosslinked efficiently to CREB or JUN proteins with K2PtCl4. The binding of TRE to CREB is not strong enough for effective detection by gel-shift assays, but the TRE-CREB complex is crosslinked efficiently by K2PtCl4 and can then readily be detected.

NASA Discipline Exobiology↗

NASA NTRS2001

Evolution of hematopoiesis: Three members of the PU.1 transcription factor family in a cartilaginous fish, Raja eglanteria

T lymphocytes and B lymphocytes are present in jawed vertebrates, including cartilaginous fishes, but not in jawless vertebrates or invertebrates. The origins of these lineages may be understood in terms of evolutionary changes in the structure and regulation of transcription factors that control lymphocyte development, such as PU.1. The identification and characterization of three members of the PU.1 family of transcription factors in a cartilaginous fish, Raja eglanteria, are described here. Two of these genes are orthologs of mammalian PU.1 and Spi-C, respectively, whereas the third gene, Spi-D, is a different family member. In addition, a PU.1-like gene has been identified in a jawless vertebrate, Petromyzon marinus (sea lamprey). Both DNA-binding and transactivation domains are highly conserved between mammalian and skate PU.1, in marked contrast to lamprey Spi, in which similarity is evident only in the DNA-binding domain. Phylogenetic analysis of sequence data suggests that the appearance of Spi-C may predate the divergence of the jawed and jawless vertebrates and that Spi-D arose before the divergence of the cartilaginous fish from the lineage leading to the mammals. The tissue-specific expression patterns of skate PU.1 and Spi-C suggest that these genes share regulatory as well as structural properties with their mammalian orthologs.

NASA Discipline Evolutionary Biology↗