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All sourcesNASA NTRSDOE OSTI
DOE OSTI2026

Convergent evolution of aerobic fermentation through divergent mechanisms acting on the same key glycolytic genes

Expression profiling by high throughput sequencing The model yeast Saccharomyces cerevisiae has evolved aerobic fermentation in part through a whole genome duplication and subsequent retention of glycolytic genes. To identify independent yeast lineages with a high glycolytic rate phenotype we developed an assay measuring extracellular acidification rates (ECAR) to evaluate the glycolytic rates across diverse yeast species. This assay identified a novel group of yeasts in the genus Saturnispora with rapid glycolytic rates. Through comparative approaches, we found that several glycolytic genes encoding hexokinases and enzymes in lower glycolysis had higher expression and modifications in promoter sequences in rapid ECAR species. Intriguingly, many of the upregulated genes are the same genes that are duplicated in S. cerevisiae. When the transcription factor required for their activation was deleted, the mutants had a slow glycolytic rate and more respiratory phenotype. This work shows how the aerobic fermentation phenotype has convergently evolved in Saturnispora through transcriptional rewiring. This divergent mechanism nevertheless impacts the same glycolytic genes, which suggests that there are evolutionary constraints on how aerobic fermentation can arise.

Horianopoulos, Linda C. [Department of Food Scienc↗
DOE OSTI2025

A haplotype-resolved reference genome for Eucalyptus grandis

Eucalyptus grandis is a hardwood tree used worldwide as pure species or hybrid partner to breed fast-growing plantation forestry crops that serve as feedstocks of timber and lignocellulosic biomass for pulp, paper, biomaterials, and biorefinery products. The current v2.0 genome reference for the species served as the first reference for the genus and has helped drive the development of molecular breeding tools for eucalypts. Using PacBio HiFi long reads and Omni-C proximity ligation sequencing, we produced an improved, haplotype-phased assembly (v4.0) for TAG0014, an early-generation selection of E. grandis. The 2 haplotypes are 571 Mbp (HAP1) and 552 Mbp (HAP2) in size and consist of 37 and 46 contigs scaffolded onto 11 chromosomes (contig N50 of 28.9 and 16.7 Mbp), respectively. These haplotype assemblies are 70-90 Mbp smaller than the diploid v2.0 assembly but capture all except one of the 22 telomeres, suggesting that substantial redundant sequence was included in the previous assembly. A total of 35,929 (HAP1) and 35,583 (HAP2) gene models were annotated, of which 438 and 472 contain long introns (>10 kbp) in gene models previously (v2.0) identified as multiple smaller genes. These and other improvements have increased gene annotation completeness levels from 93.8 to 99.4% in the v4.0 assembly. We found that 6,493 and 6,346 genes are within tandem duplicate arrays (HAP1 and HAP2, respectively, 18.4 and 17.8% of the total) and >43.8% of the haplotype assemblies consists of repeat elements. Analysis of synteny between the haplotypes and the E. grandis v2.0 reference genome revealed extensive regions of collinearity, but also some major rearrangements, and provided a preview of population and pangenome variation in the species.

Lötter, Anneri↗
DOE OSTI2025

A Chromosome-Scale Genome Assembly of the Flax Rust Fungus Reveals the Two Unusually Large Effector Proteins, AvrM3 and AvrN

Rust fungi comprise thousands of species, many of which cause disease on important crop plants. The flax rust fungus Melampsora lini has been a model species for the genetic dissection of plant immunity since the 1940s; however, the highly fragmented and incomplete reference genome has so far hindered progress in effector gene discovery. Here, we generated a fully phased, chromosome-scale assembly of the two nuclear genomes of M. lini strain CH5, resolving an additional 320 Mbp of the sequence. The 482-Mbp dikaryotic genome is at least 79% repetitive, with a large proportion (approximately 40%) of the genome comprising young, highly similar transposable elements. The assembly resolves the known effector gene loci, some of which carry complex duplications that were collapsed in the previous assembly. Using a genetic map followed by manual correction of gene models, we identified the AvrM3 and AvrN genes, which encode unusually large fungal effector proteins and trigger defense responses when co-expressed with the corresponding resistance genes. We located the genes linked to the tetrapolar mating system on chromosomes 4 and 9, but in contrast to the cereal rusts that have one pheromone receptor gene per haplotype, in flax rust, three pheromone receptor genes were found, with two of them closely linked on one haplotype. Taken together, we show that a high-quality assembly is crucial for resolving complex gene loci, and given the increasing number of fungal effectors of large size, the commonly applied criterion for effector candidates of being small proteins needs to be reconsidered.

Melampsora↗
NASA NTRS2003

Differential mesodermal expression of two amphioxus MyoD family members (AmphiMRF1 and AmphiMRF2)

To explore the evolution of myogenic regulatory factors in chordates, we isolated two MyoD family genes (AmphiMRF1 and AmphiMRF2) from amphioxus. AmphiMRF1 is first expressed at the late gastrula in the paraxial mesoderm. As the first somites form, expression is restricted to their myotomal region. In the early larva, expression is strongest in the most anterior and most posterior somites. AmphiMRF2 transcription begins at mid/late gastrula in the paraxial mesoderm, but never spreads into its most anterior region. Through much of the neurula stage, AmphiMRF2 expression is strong in the myotomal region of all somites except the most anterior pair; by late neurula expression is downregulated except in the most posterior somites forming just rostral to the tail bud. These two MRF genes of amphioxus have partly overlapping patterns of mesodermal expression and evidently duplicated independent of the diversification of the vertebrate MRF family.

NASA Discipline Evolutionary Biology↗
DOE OSTI2026

BSMV-mediated genome editing exhibits host-specific heritability: germline transmission in barley and somatic edits in Nicotiana benthamiana

Plant RNA virus–mediated guide RNA (gRNA) delivery represents a transformative advance in genome editing technologies. Unlike conventional transformation methods that rely on labor-intensive tissue culture and regeneration for each individual gRNA delivery, viral vectors can rapidly and systemically transmit gRNAs into pre-established Cas-expressing plants, providing an accelerated route for functional genomics and trait discovery directly in planta . However, key design parameters, including subgenomic promoter choice, transcript architecture, and their effects on viral fitness and editing outcomes, remain to be elucidated for most viral platforms. We developed five Barley stripe mosaic virus (BSMV) vectors, each with distinct subgenomic promoter elements to drive single gRNA expression. These were initially evaluated in Cas9-expressing transgenic Nicotiana benthamiana plants targeting the Phytoene desaturase ( PDS ) gene to compare their editing efficiencies. Single gRNAs expressed under the duplicated γb subgenomic promoter or when fused directly to the γb genome achieved the highest mutation frequencies (up to 90% at 60 days post-inoculation), whereas β1- and β2-driven sgRNAs produced delayed and reduced editing. Thus, promoter selection critically determines gRNA accumulation and the efficacy of BSMV-mediated genome editing. The top-performing design was then applied to Cas9-expressing barley ( Hordeum vulgare ) targeting HvCMF7 (conferring green-white variegation) and HvGW2.1 (impacts grain width and weight). BSMV spread systemically throughout barley, inducing somatic and heritable mutations at frequencies up to 100%, with virus-free edited progeny. In contrast, despite robust somatic editing in N. benthamiana, no heritable mutations were detected indicating species-dependent limitations in germline transmission. Our systematic comparison of subgenomic promoter architectures establishes clear design principles for optimizing viral vector–mediated delivery. Promoter choice and transcript structure critically shape editing efficiency and viral stability. The host-specific boundary for germline editing, defined by efficient heritable editing in barley but not N. benthamiana , highlights where BSMV offers advantages and where alternative vectors or hybrid strategies are required, guiding rational platform selection for diverse crop species and applications. Collectively, these findings establish BSMV as a promising next-generation vector for rapid, tissue culture–free, and transformation-independent genome editing in cereals and other recalcitrant monocots.

barley↗
DOE OSTI2024

Adaptive laboratory evolution and genetic engineering improved terephthalate utilization in Pseudomonas putida KT2440

Poly(ethylene terephthalate) (PET) is one of the most ubiquitous plastics and can be depolymerized through biological and chemo-catalytic routes to its constituent monomers, terephthalic acid (TPA) and ethylene glycol (EG). TPA and EG can be re-synthesized into PET for closed-loop recycling or microbially converted into higher-value products for open-loop recycling. Here, in this study, we expand on our previous efforts engineering and applying Pseudomonas putida KT2440 for PET conversion by employing adaptive laboratory evolution (ALE) to improve TPA catabolism. Three P. putida strains with varying degrees of metabolic engineering for EG catabolism underwent an automation-enabled ALE campaign on TPA, a TPA and EG mixture, and glucose as a control. ALE increased the growth rate on TPA and TPA-EG mixtures by 4.1- and 3.5-fold, respectively, in approximately 350 generations. Evolved isolates were collected at the midpoints and endpoints of 39 independent ALE experiments, and growth rates were increased by 0.15 and 0.20 h -1 on TPA and a TPA-EG, respectively, in the best performing isolates. Whole-genome re-sequencing identified multiple converged mutations, including loss-of-function mutations to global regulators gacS, gacA, and turA along with large duplication and intergenic deletion events that impacted the heterologously-expressed tphAB II catabolic genes. Reverse engineering of these targets confirmed causality, and a strain with all three regulators deleted and second copies of tphAB II and tpaK displayed improved TPA utilization compared to the base strain. Taken together, an iterative strain engineering process involving heterologous pathway engineering, ALE, whole genome sequencing, and genome editing identified five genetic interventions that improve P. putida growth on TPA, aimed at developing enhanced whole-cell biocatalysts for PET upcycling.

36 MATERIALS SCIENCE↗
NASA NTRS1994

Transcription factor IID in the Archaea: sequences in the Thermococcus celer genome would encode a product closely related to the TATA-binding protein of eukaryotes

The first step in transcription initiation in eukaryotes is mediated by the TATA-binding protein, a subunit of the transcription factor IID complex. We have cloned and sequenced the gene for a presumptive homolog of this eukaryotic protein from Thermococcus celer, a member of the Archaea (formerly archaebacteria). The protein encoded by the archaeal gene is a tandem repeat of a conserved domain, corresponding to the repeated domain in its eukaryotic counterparts. Molecular phylogenetic analyses of the two halves of the repeat are consistent with the duplication occurring before the divergence of the archael and eukaryotic domains. In conjunction with previous observations of similarity in RNA polymerase subunit composition and sequences and the finding of a transcription factor IIB-like sequence in Pyrococcus woesei (a relative of T. celer) it appears that major features of the eukaryotic transcription apparatus were well-established before the origin of eukaryotic cellular organization. The divergence between the two halves of the archael protein is less than that between the halves of the individual eukaryotic sequences, indicating that the average rate of sequence change in the archael protein has been less than in its eukaryotic counterparts. To the extent that this lower rate applies to the genome as a whole, a clearer picture of the early genes (and gene families) that gave rise to present-day genomes is more apt to emerge from the study of sequences from the Archaea than from the corresponding sequences from eukaryotes.

NASA Discipline Exobiology↗
NASA NTRS2000

Spaceflight effects on cultured embryonic chick bone cells

A model calcifying system of primary osteoblast cell cultures derived from normal embryonic chicken calvaria has been flown aboard the shuttle, Endeavour, during the National Aeronautics and Space Administration (NASA) mission STS-59 (April 9-20, 1994) to characterize unloading and other spaceflight effects on the bone cells. Aliquots of cells (approximately 7 x 10(6)) grown in Dulbecco's modified Eagle's medium (DMEM) + 10% fetal bovine serum (FBS) were mixed with microcarrier beads, inoculated into cartridge culture units of artificial hollow fiber capillaries, and carried on the shuttle. To promote cell differentiation, cartridge media were supplemented with 12.5 microg/ml ascorbate and 10 mM beta-glycerophosphate for varying time periods before and during flight. Four cartridges contained cells from 17-day-old embryos grown for 5 days in the presence of ascorbate prior to launch (defined as flight cells committed to the osteoblastic lineage) and four cartridges supported cells from 14-day-old embryos grown for 10 days with ascorbate before launch (uncommitted flight cells). Eight cartridges prepared in the same manner were maintained under normal gravity throughout the flight (control cells) and four additional identical cartridges under normal gravity were terminated on the day of launch (basal cells). From shuttle launch to landing, all cartridges were contained in closed hardware units maintaining 5% CO2, 37 degrees C, and media delivery at a rate of approximately 1.5 ml/6 h. During day 3 and day 5 of flight, duplicate aliquots of conditioned media and accumulated cell products were collected in both the flight and the control hardware units. At the mission end, comparisons among flight, basal, and control samples were made in cell metabolism, gene expression for type I collagen and osteocalcin, and ultrastructure. Both committed and uncommitted flight cells were metabolically active, as measured by glucose uptake and lactate production, at approximately the same statistical levels as control counterparts. Flight cells elaborated a less extensive extracellular matrix, evidenced by a reduced collagen gene expression and collagen protein appearance compared with controls. Osteocalcin was expressed by all cells, a result indicating progressive differentiation of both flight and control osteoblasts, but its message levels also were reduced in flight cells compared with ground samples. This finding suggested that osteoblasts subjected to flight followed a slower progression toward a differentiated function. The summary of data indicates that spaceflight, including microgravity exposure, demonstrably affects bone cells by down-regulating type I collagen and osteocalcin gene expression and thereby inhibiting expression of the osteogenic phenotype notably by committed osteoblasts. The information is important for insight into the response of bone cells to changes of gravity and of force in general.

Non-NASA Center↗
DOE OSTI2025

Convergent evolution of NFP -facilitated root nodule symbiosis

The origin and phylogenetic distribution of symbiotic associations between nodulating angiosperms and nitrogen-fixing bacteria have long intrigued biologists. Recent comparative evolutionary analyses have yielded alternative hypotheses: a multistep pathway of independent gains and losses of root nodule symbiosis vs. a single gain followed by numerous losses. A detailed reconstruction of the history of genes involved in signaling between nitrogen-fixing bacteria and potential hosts, particularly lipo-chitooligosaccharide (LCO) signaling, is needed to distinguish between these hypotheses. LCO recognition by plants involves the Nod Factor Perception ( NFP ) gene family; in the legume model Medicago truncatula (Fabales), MtNFP is essential for establishing rhizobial symbiosis. Here, we document convergent evolution of NFP , indicating multiple origins of LCO-driven symbiosis. In contrast to previous models that explain the recruitment of NFP via a single duplication in the ancestor of the nitrogen-fixing clade, our phylogenomic and synteny results suggest this duplication does not span the entire clade. Tandem duplication in a common ancestor of Cucurbitales and Rosales resulted in the NFP1 and NFP2 groups. In contrast, the phylogenetically closest paralog of MtNFP is MtLYR1 , located on a different chromosome within a large syntenic block. All available data indicate that a large-scale duplication resulted in MtNFP and MtLYR1 , likely corresponding to a whole-genome duplication in an ancestor of subfamily Papilionoideae of Fabaceae. We show that MtNFP and the NFP2 -like group are not orthologous, indicating multiple independent gains of NFP -based LCO signaling. This molecular convergence provides a possible mechanism for multiple gains of root nodule symbiosis across the nitrogen-fixing clade.

LCO signaling↗
DOE OSTI2025

Tonoplast Sucrose Transporter SUT4-Dependent Sugar Partitioning Modulates Phenological Transitions and Reproductive Success in Poplar

Climate uncertainty is intensifying the need for greater plasticity in carbohydrate reserve utilization to support winter survival and spring growth in woody perennials. In poplar, the single-copy SUT4, which encodes a tonoplast-localized sucrose transporter, and the SUT5/SUT6 genome duplicates, which encode plasma membrane-localized transporters, are expressed year-round, with SUT4 showing the highest expression during cool seasons. Given its role in vacuolar sucrose efflux and winter-predominant expression, SUT4 may play a key role in modulating seasonal carbohydrate dynamics. While SUT4-knockdown and knockout effects have been studied under greenhouse conditions, their impact under field conditions remains unexplored. Here, we report a field-based study comparing CRISPR knockout mutants of winter-expressed SUT4 and SUT5/SUT6 in Populus tremula x alba. We show that sut4, but not sut5/6, mutants exhibited earlier autumn leaf senescence, delayed spring bud flush, reduced stem growth, and altered sugar partitioning in winter xylem and bark relative to controls. After 2 years in the field, all genotypes flowered before leaf flush in early spring; however, sut4 mutants produced sterile ovules despite developing normal-looking catkins. Metabolic profiling revealed disrupted sucrose and raffinose dynamics in elongating sut4 catkins. This was accompanied by transcriptomic signatures of elevated stress and downregulation of proanthocyanidin biosynthesis and circadian clock genes. These findings highlight the critical role of SUT4 in coordinating sugar allocation, stress responses, and seasonal development in poplar.

09 BIOMASS FUELS↗
NASA NTRS1998

The Toll pathway is required in the epidermis for muscle development in the Drosophila embryo

The Toll signaling pathway functions in several Drosophila processes, including dorsal-ventral pattern formation and the immune response. Here, we demonstrate that this pathway is required in the epidermis for proper muscle development. Previously, we showed that the zygotic Toll protein is necessary for normal muscle development; in the absence of zygotic Toll, close to 50% of hemisegments have muscle patterning defects consisting of missing, duplicated and misinserted muscle fibers (Halfon, M.S., Hashimoto, C., and Keshishian, H., Dev. Biol. 169, 151-167, 1995). We have now also analyzed the requirements for easter, spatzle, tube, and pelle, all of which function in the Toll-mediated dorsal-ventral patterning pathway. We find that spatzle, tube, and pelle, but not easter, are necessary for muscle development. Mutations in these genes give a phenotype identical to that seen in Toll mutants, suggesting that elements of the same pathway used for Toll signaling in dorsal-ventral development are used during muscle development. By expressing the Toll cDNA under the control of distinct Toll enhancer elements in Toll mutant flies, we have examined the spatial requirements for Toll expression during muscle development. Expression of Toll in a subset of epidermal cells that includes the epidermal muscle attachment cells, but not Toll expression in the musculature, is necessary for proper muscle development. Our results suggest that signals received by the epidermis early during muscle development are an important part of the muscle patterning process.

Non-NASA Center↗
NASA NTRS2002

NRSF causes cAMP-sensitive suppression of sodium current in cultured hippocampal neurons

The neuron restrictive silencer factor (NRSF/REST) has been shown to bind to the promoters of many neuron-specific genes and is able to suppress transcription of Na(+) channels in PC12 cells, although its functional effect in terminally differentiated neurons is unknown. We constructed lentiviral vectors to express NRSF as a bicistronic message with green fluorescent protein (GFP) and followed infected hippocampal neurons in culture over a period of 1-2 wk. NRSF-expressing neurons showed a time-dependent suppression of Na(+) channel function as measured by whole cell electrophysiology. Suppression was reversed or prevented by the addition of membrane-permeable cAMP analogues and enhanced by cAMP antagonists but not affected by increasing protein expression with a viral enhancer. Secondary effects, including altered sensitivity to glutamate and GABA and reduced outward K(+) currents, were duplicated by culturing GFP-infected control neurons in TTX. The striking similarity of the phenotypes makes NRSF potentially useful as a genetic "silencer" and also suggests avenues of further exploration that may elucidate the transcription factor's in vivo role in neuronal plasticity.

Hippocampus/cytology/metabolism↗
NASA NTRS2014

Biosentinel: Developing a Space Radiation Biosensor

Ionizing radiation presents a major challenge to human exploration and long-term residence in space. The deep-space radiation spectrum includes highly energetic particles that generate double strand breaks (DSBs), deleterious DNA lesions that are usually repaired without errors via homologous recombination (HR), a conserved pathway in all eukaryotes. While progress identifying and characterizing biological radiation effects using Earth-based facilities has been significant, no terrestrial source duplicates the unique space radiation environment.We are developing a biosensor-based nanosatellite to fly aboard NASAs Space Launch System Exploration Mission 1, expected to launch in 2017 and reach a 1AU (astronomic unit) heliocentric orbit. Our biosensor (called BioSentinel) uses the yeast S. cerevisiae to measure DSBs in response to ambient space radiation. The BioSentinel strain contains engineered genetic defects that prevent growth until and unless a radiation-induced DSB near a reporter gene activates the yeasts HR repair mechanisms. Thus, culture growth and metabolic activity directly indicate a successful DSB-and-repair event. In parallel, HR-defective and wild type strains will provide survival data. Desiccated cells will be carried within independent culture microwells, built into 96-well microfluidic cards. Each microwell set will be activated by media addition at different time points over 18 months, and cell growth will be tracked continuously via optical density. One reserve set will be activated only in the occurrence of a solar particle event. Biological measurements will be compared to data provided by onboard physical dosimeters and to Earth-based experiments.BioSentinel will conduct the first study of biological response to space radiation outside Low Earth Orbit in over 40 years. BioSentinel will thus address strategic knowledge gaps related to the biological effects of space radiation and will provide an adaptable platform to perform human-relevant measurements in multiple space environments. We hope that it can therefore be used on the ISS, on and around other planetary bodies as well as other exploration platforms as a self-contained system that will allow us to compare and calibrate different radiation environments.BioSentinels results will be critical for improving interpretation of the effects of space radiation exposure, and for reducing the risk associated with long-term human exploration.

space radiation↗
NASA NTRS1998

The prokaryote-to-eukaryote transition reflected in the evolution of the V/F/A-ATPase catalytic and proteolipid subunits

Changes in the primary and quarternary structure of vacuolar and archaeal type ATPases that accompany the prokaryote-to-eukaryote transition are analyzed. The gene encoding the vacuolar-type proteolipid of the V-ATPase from Giardia lamblia is reported. Giardia has a typical vacuolar ATPase as observed from the common motifs shared between its proteolipid subunit and other eukaryotic vacuolar ATPases, suggesting that the former enzyme works as a hydrolase in this primitive eukaryote. The phylogenetic analyses of the V-ATPase catalytic subunit and the front and back halves of the proteolipid subunit placed Giardia as the deepest branch within the eukaryotes. Our phylogenetic analysis indicated that at least two independent duplication and fusion events gave rise to the larger proteolipid type found in eukaryotes and in Methanococcus. The spatial distribution of the conserved residues among the vacuolar-type proteolipids suggest a zipper-type interaction among the transmembrane helices and surrounding subunits of the V-ATPase complex. Important residues involved in the function of the F-ATP synthase proteolipid have been replaced during evolution in the V-proteolipid, but in some cases retained in the archaeal A-ATPase. Their possible implication in the evolution of V/F/A-ATPases is discussed.

NASA Discipline Exobiology↗
NASA NTRS1990

A study of murine bone marrow cells cultured in bioreactors which create an environment which simulated microgravity

Previous research indicated that mouse bone marrow cells could be grown in conditions of simulated microgravity. This environment was created in rotating bioreactor vessels. On three attempts mouse cells were grown successfully in the vessels. The cells reached a stage where the concentrations were doubling daily. Phenotypic analysis using a panel of monoclonal antibodies indicated that the cell were hematopoietic pluripotent stem cells. One unsuccessful attempt was made to reestablish the immune system in immunocompromised mice using these cells. Since last summer, several unsuccessful attempts were made to duplicate these results. It was determined by electron microscopy that the cells successfully grown in 1989 contained virus particles. It was suggested that these virally parasitized cells had been immortalized. The work of this summer is a continuation of efforts to grow mouse bone marrow in these vessels. A number of variations of the protocol were introduced. Certified pathogen free mice were used in the repeat experiments. In some attempts the medium of last summer was used; in others Dexture Culture Medium containing Iscove's Medium supplemented with 20 percent horse serum and 10-6 M hydrocortisone. Efforts this summer were directed solely to repeating the work of last summer. Plans were made for investigations if stem cells were isolated. Immortalization of the undifferentiated stem cell would be attempted by transfection with an oncogenic vector. Selective differentiation would be induced in the stem cell line by growing it with known growth factors and immune response modulators. Interest is in identifying any surface antigens unique to stem cells that would help in their characterization. Another goal was to search for markers on stem cells that would distinguish them from stem cells committed to a particular lineage. If the undifferentiated hematopoietic stem cell was obtained, the pathways that would terminally convert it to myeloid, lyphoid, erythroid, or other cell lines would be studied. Transfection with a known gene would be attempted and then conversion to a terminally identifiable cell.

Lawless, Brother Desales↗
NASA NTRS2000

Functional conservation of atonal and Math1 in the CNS and PNS

To determine the extent to which atonal and its mouse homolog Math1 exhibit functional conservation, we inserted (beta)-galactosidase (lacZ) into the Math1 locus and analyzed its expression, evaluated consequences of loss of Math1 function, and expressed Math1 in atonal mutant flies. lacZ under the control of Math1 regulatory elements duplicated the previously known expression pattern of Math1 in the CNS (i.e., the neural tube, dorsal spinal cord, brainstem, and cerebellar external granule neurons) but also revealed new sites of expression: PNS mechanoreceptors (inner ear hair cells and Merkel cells) and articular chondrocytes. Expressing Math1 induced ectopic chordotonal organs (CHOs) in wild-type flies and partially rescued CHO loss in atonal mutant embryos. These data demonstrate that both the mouse and fly homologs encode lineage identity information and, more interestingly, that some of the cells dependent on this information serve similar mechanoreceptor functions.

NASA Discipline Developmental Biology↗
NASA NTRS2001

Expression of the Acyl-Coenzyme A: Cholesterol Acyltransferase GFP Fusion Protein in Sf21 Insect Cells

The enzyme acyl-coenzyme A:cholesterol acyltransferase (ACAT) is an important contributor to the pathological expression of plaque leading to artherosclerosis n a major health problem. Adequate knowledge of the structure of this protein will enable pharmaceutical companies to design drugs specific to the enzyme. ACAT is a membrane protein located in the endoplasmic reticulum.t The protein has never been purified to homogeneity.T.Y. Chang's laboratory at Dartmouth College provided a 4-kb cDNA clone (K1) coding for a structural gene of the protein. We have modified the gene sequence and inserted the cDNA into the BioGreen His Baculovirus transfer vector. This was successfully expressed in Sf2l insect cells as a GFP-labeled ACAT protein. The advantage to this ACAT-GFP fusion protein (abbreviated GCAT) is that one can easily monitor its expression as a function of GFP excitation at 395 nm and emission at 509 nm. Moreover, the fusion protein GCAT can be detected on Western blots with the use of commercially available GFP antibodies. Antibodies against ACAT are not readily available. The presence of the 6xHis tag in the transfer vector facilitates purification of the recombinant protein since 6xHis fusion proteins bind with high affinity to Ni-NTA agarose. Obtaining highly pure protein in large quantities is essential for subsequent crystallization. The purified GCAT fusion protein can readily be cleaved into distinct GFP and ACAT proteins in the presence of thrombin. Thrombin digests the 6xHis tag linking the two protein sequences. Preliminary experiments have indicated that both GCAT and ACAT are expressed as functional proteins. The ultimate aim is to obtain large quantities of the ACAT protein in pure and functional form appropriate for protein crystal growth. Determining protein structure is the key to the design and development of effective drugs. X-ray analysis requires large homogeneous crystals that are difficult to obtain in the gravity environment of earth. Protein crystals grown in microgravity are often larger and have fewer defects than those grown on earth. The analysis of higher quality space-grown crystals will assist in structure-based drug design. We have successfully grown GCAT-infected Sf21 cells in both adhesion and suspension cultures. Expression levels of GCAT in cell lines such as Sf9 and High Five appear to be reduced. We intend to replicate GCAT expression in all three cell lines using the NASA rotating wall bioreactor which effectively duplicates a microgravity environment. The bioreactor itself could be launched to study the expression of the GFP and GCAT proteins in the actual microgravity environment achieved in orbit.

Mahtani, H. K.↗