SEARCH · Search NASA

Results for “gene expression analysis”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

SourceResearch connectionFrom yearThrough year

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 199 records · Page 11

All sources NASA NTRS DOE OSTI

NASA NTRS1995

Sequence, molecular properties, and chromosomal mapping of mouse lumican

PURPOSE. Lumican is a major proteoglycan of vertebrate cornea. This study characterizes mouse lumican, its molecular form, cDNA sequence, and chromosomal localization. METHODS. Lumican sequence was determined from cDNA clones selected from a mouse corneal cDNA expression library using a bovine lumican cDNA probe. Tissue expression and size of lumican mRNA were determined using Northern hybridization. Glycosidase digestion followed by Western blot analysis provided characterization of molecular properties of purified mouse corneal lumican. Chromosomal mapping of the lumican gene (Lcn) used Southern hybridization of a panel of genomic DNAs from an interspecific murine backcross. RESULTS. Mouse lumican is a 338-amino acid protein with high-sequence identity to bovine and chicken lumican proteins. The N-terminus of the lumican protein contains consensus sequences for tyrosine sulfation. A 1.9-kb lumican mRNA is present in cornea and several other tissues. Antibody against bovine lumican reacted with recombinant mouse lumican expressed in Escherichia coli and also detected high molecular weight proteoglycans in extracts of mouse cornea. Keratanase digestion of corneal proteoglycans released lumican protein, demonstrating the presence of sulfated keratan sulfate chains on mouse corneal lumican in vivo. The lumican gene (Lcn) was mapped to the distal region of mouse chromosome 10. The Lcn map site is in the region of a previously identified developmental mutant, eye blebs, affecting corneal morphology. CONCLUSIONS. This study demonstrates sulfated keratan sulfate proteoglycan in mouse cornea and describes the tools (antibodies and cDNA) necessary to investigate the functional role of this important corneal molecule using naturally occurring and induced mutants of the murine lumican gene.

NASA Discipline Cell Biology↗

NASA NTRS2001

Shear stress reduces protease activated receptor-1 expression in human endothelial cells

Shear stress has been shown to regulate several genes involved in the thrombotic and proliferative functions of endothelial cells. Thrombin receptor (protease-activated receptor-1: PAR-1) increases at sites of vascular injury, which suggests an important role for PAR-1 in vascular diseases. However, the effect of shear stress on PAR-1 expression has not been previously studied. This work investigates effects of shear stress on PAR-1 gene expression in both human umbilical vein endothelial cells (HUVECs) and microvascular endothelial cells (HMECs). Cells were exposed to different shear stresses using a parallel plate flow system. Northern blot and flow cytometry analysis showed that shear stress down-regulated PAR-1 messenger RNA (mRNA) and protein levels in both HUVECs and HMECs but with different thresholds. Furthermore, shear-reduced PAR-1 mRNA was due to a decrease of transcription rate, not increased mRNA degradation. Postshear stress release of endothelin-1 in response to thrombin was reduced in HUVECs and HMECs. Moreover, inhibitors of potential signaling pathways applied during shear stress indicated mediation of the shear-decreased PAR-1 expression by protein kinases. In conclusion, shear stress exposure reduces PAR-1 gene expression in HMECs and HUVECs through a mechanism dependent in part on protein kinases, leading to altered endothelial cell functional responses to thrombin.

Non-NASA Center↗

NASA NTRS2014

Gene Expression Profiling of Lung Tissue of Rats Exposed to Lunar Dust Particles

The purpose of the study is to analyze the dynamics of global gene expression changes in the lung tissue of rats exposed to lunar dust particles. Multiple pathways and transcription factors were identified using the Ingenuity Pathway Analysis tool, showing the potential networks of these signaling regulations involved in lunar dust‐induced prolonged proflammatory response and toxicity. The data presented in this study, for the first time, explores the molecular mechanisms of lunar dust induced toxicity. This work contributes not only to the risk assessment for future space exploration, but also to the understanding of the dust‐induced toxicity to humans on earth.

Zhang, Ye↗

NASA NTRS2001

Atrogin-1, a muscle-specific F-box protein highly expressed during muscle atrophy

Muscle wasting is a debilitating consequence of fasting, inactivity, cancer, and other systemic diseases that results primarily from accelerated protein degradation by the ubiquitin-proteasome pathway. To identify key factors in this process, we have used cDNA microarrays to compare normal and atrophying muscles and found a unique gene fragment that is induced more than ninefold in muscles of fasted mice. We cloned this gene, which is expressed specifically in striated muscles. Because this mRNA also markedly increases in muscles atrophying because of diabetes, cancer, and renal failure, we named it atrogin-1. It contains a functional F-box domain that binds to Skp1 and thereby to Roc1 and Cul1, the other components of SCF-type Ub-protein ligases (E3s), as well as a nuclear localization sequence and PDZ-binding domain. On fasting, atrogin-1 mRNA levels increase specifically in skeletal muscle and before atrophy occurs. Atrogin-1 is one of the few examples of an F-box protein or Ub-protein ligase (E3) expressed in a tissue-specific manner and appears to be a critical component in the enhanced proteolysis leading to muscle atrophy in diverse diseases.

NASA Discipline Musculoskeletal↗

NASA NTRS2003

Conservation of Endo16 expression in sea urchins despite evolutionary divergence in both cis and trans-acting components of transcriptional regulation

Evolutionary changes in transcriptional regulation undoubtedly play an important role in creating morphological diversity. However, there is little information about the evolutionary dynamics of cis-regulatory sequences. This study examines the functional consequence of evolutionary changes in the Endo16 promoter of sea urchins. The Endo16 gene encodes a large extracellular protein that is expressed in the endoderm and may play a role in cell adhesion. Its promoter has been characterized in exceptional detail in the purple sea urchin, Strongylocentrotus purpuratus. We have characterized the structure and function of the Endo16 promoter from a second sea urchin species, Lytechinus variegatus. The Endo16 promoter sequences have evolved in a strongly mosaic manner since these species diverged approximately 35 million years ago: the most proximal region (module A) is conserved, but the remaining modules (B-G) are unalignable. Despite extensive divergence in promoter sequences, the pattern of Endo16 transcription is largely conserved during embryonic and larval development. Transient expression assays demonstrate that 2.2 kb of upstream sequence in either species is sufficient to drive GFP reporter expression that correctly mimics this pattern of Endo16 transcription. Reciprocal cross-species transient expression assays imply that changes have also evolved in the set of transcription factors that interact with the Endo16 promoter. Taken together, these results suggest that stabilizing selection on the transcriptional output may have operated to maintain a similar pattern of Endo16 expression in S. purpuratus and L. variegatus, despite dramatic divergence in promoter sequence and mechanisms of transcriptional regulation.

NASA Discipline Evolutionary Biology↗

NASA NTRS2010

Immune System Dysregulation and Herpesvirus Reactivation Persist During Long-Duration Spaceflight

Background: Immunity, latent herpesvirus reactivation, physiological stress and circadian rhythms were assessed during six month spaceflight onboard ISS. Blood and saliva samples were collected early, mid and late in-flight and returned for immediate analysis. Mid-point study data (10 of 17 planned subjects) will be presented. Results: Some shifts in leukocyte distribution occurred during flight, including alterations in CD8+ T cell maturation. General T cell function was consistently reduced early in-flight. Levels CD8+/IFNg+ producing T cells were depressed early in-flight, and immediately upon landing. Persistent mitogen-dependant reductions were observed in IFNg, IL-17a, IL-10, TNFa and IL-6 production. Monocyte production of IL-10 was reduced, whereas IL-8 levels were increased. Levels of mRNA for the TNFa, IL-6 and IFNg were transiently elevated early in-flight, and the dynamics of TNF and IL-6 gene expression were somewhat antagonistic to their corresponding receptors during flight. The number of virus-specific CD8+ T-cells was measured using MHC tetramers, while their function was measured using intracellular cytokine analysis following peptide stimulation. Both the number and function of EBV-specific cells decreased during flight as compared to preflight levels. The number of CMV-specific T-cells generally increased as the mission progressed while their function was variable. Viral (EBV) load in blood was elevated postflight. Anti-EBV VCA antibodies were significantly elevated by R+0; anti-EA antibodies were not significantly elevated at landing; and anti-CMV antibodies were somewhat elevated during flight. Higher levels of salivary EBV DNA were found during flight. VZV DNA reactivation occurred in ~50 % of astronauts during flight, continuing for up to 30 days post-flight. CMV was shed in 35 % the in-flight and 30% of postflight urine samples of the crewmembers. There was generally a higher level of cortisol as measured in urine and saliva in the astronauts during flight, but plasma cortisol was relatively unchanged during flight. Circadian rhythm of salivary cortisol was altered during flight. Conclusion. Some alterations in immunity do not resolve during six month spaceflight, consequentially resulting in persistent herpesvirus reactivation. Ongoing immune dysregulation may represent specific clinical risks for exploration-class space missions.

Crucian, B. E.↗

NASA NTRS1998

PCR cloning and characterization of multiple ADP-glucose pyrophosphorylase cDNAs from tomato

Four ADP-glucose pyrophosphorylase (AGP) cDNAs were cloned from tomato fruit and leaves by the PCR techniques. Three of them (agp S1, agp S2, and agp S3) encode the large subunit of AGP, the fourth one (agp B) encodes the small subunit. The deduced amino acid sequences of the cDNAs show very high identities (96-98%) to the corresponding potato AGP isoforms, although there are major differences in tissue expression profiles. All four tomato AGP transcripts were detected in fruit and leaves; the predominant ones in fruit are agp B and agp S1, whereas in leaves they are agp B and agp S3. Genomic southern analysis suggests that the four AGP transcripts are encoded by distinct genes.

Non-NASA Center↗

NASA NTRS2003

Dual reporter transgene driven by 2.3Col1a1 promoter is active in differentiated osteoblasts

AIM: As quantitative and spatial analyses of promoter reporter constructs are not easily performed in intact bone, we designed a reporter gene specific to bone, which could be analyzed both visually and quantitatively by using chloramphenicol acetyltransferase (CAT) and a cyan version of green fluorescent protein (GFPcyan), driven by a 2.3-kb fragment of the rat collagen promoter (Col2.3). METHODS: The construct Col2.3CATiresGFPcyan was used for generating transgenic mice. Quantitative measurement of promoter activity was performed by CAT analysis of different tissues derived from transgenic animals; localization was performed by visualized GFP in frozen bone sections. To assess transgene expression during in vitro differentiation, marrow stromal cell and neonatal calvarial osteoblast cultures were analyzed for CAT and GFP activity. RESULTS: In mice, CAT activity was detected in the calvaria, long bone, teeth, and tendon, whereas histology showed that GFP expression was limited to osteoblasts and osteocytes. In cell culture, increased activity of CAT correlated with increased differentiation, and GFP activity was restricted to mineralized nodules. CONCLUSION: The concept of a dual reporter allows a simultaneous visual and quantitative analysis of transgene activity in bone.

Non-NASA Center↗

NASA NTRSDate not supplied

Biological Insights at the Interface of Multiple Arabidopsis Legacy Datasets

The NASA GeneLab database includes an open-access collection of datasets yielded by space biology experiments. Six Gene Lab Data Sets (GLDS’s) performed in Arabidopsis were selected for analysis (7/17/44/121/205/213), all of which included transcriptome data from spaceflight and ground control environments. Hardware, ecotype, environmental conditions, and other experimental conditions varied, allowing the observations of overarching gene expression impacts of microgravity on plant life without focusing on effects of specific experimental conditions. Using GeneLab pre-processed datasets as the basis for the study, RNA microarray data were analyzed to identify genes that showed altered expression in microgravity when compared to control samples for each individual GLDS. All differentially expressed genes were compared to locate differentially expressed genes common between spaceflight experiments. The most noteworthy result is that not one gene shared differential expression among the six GLDS’s. However, gene expression was not influenced randomly by the microgravity environment, as there were several gene ontology terms that were significantly enriched across all experiments. These included 20 significantly enriched biological processes, and although the genes which enriched each term varied, there were many cases of specific genes common to clusters of multiple GLDS’s. Gene expression such as NAC92 and ERF011 or membrane structural element FFP6 provide insight and direction toward understanding the plant response to spaceflight. Characterizing these common processes and the shared differentially expressed genes has demonstrated potential targets for further study to understand and modulate the biological response of plants in microgravity. Life on Earth has never been subjected to the absence of gravity as a selective pressure, so observing how life forms react to a microgravity environment could provide insight to our shared fundamental biological processes. It is also feasible that the genetic modification of specific genes linked to the microgravity response could improve health and yield of space crops.

Joseph Emhof↗

NASA NTRSDate not supplied

Translational Line of Sight: a multi-omics longitudinal study of the murine retinal response to spaceflight hazard analogs

The space environment includes unique hazards like radiation and microgravity which adversely affect physiology and behavior of humans and rodent models. To better characterize the retinal response to spaceflight, we assessed a multi-omics NASA GeneLab dataset where 6-month-old female mice were gamma irradiated and/or hindlimb unloaded for 21 days followed by whole transcriptome shotgun sequencing (RNA-Seq) and reduced representation bisulfite sequencing (RRBS) of retina samples collected at 7 days, 1 month or 4 months post-exposure. We compared time-matched epigenomic and transcriptomic retinal profiles revealing a total of 4,178 differentially methylated loci or regions, and 457 differentially expressed genes. Highest correlation in methylation differences was seen across different conditions at the same time point (e.g., between radiation exposure and hindlimb unloaded at 7 days). Biological processes related to nucleotide metabolism were enriched in all groups with activation at 1 month and suppression at 7 days and 4 months. Genes and processes related to Notch and Wnt signaling showed alterations 4 months post-exposure. Interestingly, Notch3 and Lrg1 showed differential patterns in the NASA Twins Study in-flight samples and in response to stressors in the murine retina in the current study. A total of 23 genes were both differentially methylated and expressed, including genes involved in retinal disease or cataract development (Crybb3, Fgfr1, Pitpnm3, Sipa1l3, Sox9) and inflammatory response (B4galt6, Ppm1a, Sphk1). To our knowledge, the current multi-omics analysis is the first multi-omics study to interrogate the epigenomic and transcriptomic impacts of radiation and hindlimb unloading on the retina in isolation and in combination. The results provide an insight into the retinal response to individual spaceflight hazard analogs and their interplay at different post-exposure stages and contributes towards a mechanistic understanding of spaceflight-induced vision impairment using ground-based models.

Prachi Kothiyal↗

NASA NTRSDate not supplied

A Multi-omics Longitudinal Study of the Murine Retinal Response to Chronic Low-dose Irradiation and/or Simulated Microgravity

Prachi Kothiyal↗

NASA NTRS1995

Two cis elements collaborate to spatially repress transcription from a sea urchin promoter

The expression pattern of many territory-specific genes in metazoan embryos is maintained by an active process of negative spatial regulation. However, the mechanism of this strategy of gene regulation is not well understood in any system. Here we show that reporter constructs containing regulatory sequence for the SM30-alpha gene of Stronglyocentrotus purpuratus are expressed in a pattern congruent with that of the endogenous SM30 gene(s), largely as a result of active transcriptional repression in cell lineages in which the gene is not normally expressed. Chloramphenicol acetyl transferase assays of deletion constructs from the 2600-bp upstream region showed that repressive elements were present in the region from -1628 to -300. In situ hybridization analysis showed that the spatial fidelity of expression was severely compromised when the region from -1628 to -300 was deleted. Two highly repetitive sequence motifs, (G/A/C)CCCCT and (T/C)(T/A/C)CTTTT(T/A/C), are present in the -1628 to -300 region. Representatives of these elements were analyzed by gel mobility shift experiments and were found to interact specifically with protein in crude nuclear extracts. When oligonucleotides containing either sequence element were co-injected with a correctly regulated reporter as potential competitors, the reporter was expressed in inappropriate cells. When composite oligonucleotides, containing both sequence elements, were fused to a misregulated reporter, the expression of the reporter in inappropriate cells was suppressed. Comparison of composite oligonucleotides with oligonucleotides containing single constituent elements show that both sequence elements are required for effective spatial regulation. Thus, both individual elements are required, but only a composite element containing both elements is sufficient to function as a tissue-specific repressive element.

Non-NASA Center↗

NASA NTRS2016

Elucidating the Gravome: Quantitative Proteomic Profiling of the Response to Chronic Hypergravity in Drosophila

Altered gravity conditions, such as experienced by organisms during spaceflight, is known to cause transcriptomic and proteomic changes. We describe the proteomic changes in the whole body of adult Drosophila melanogaster (fruit fly), but focus specifically on the localized changes in the adult head in response to chronic hypergravity (3G) treatment. Canton S adult female flies (2-3 days old) were exposed to chronic hypergravity for 9 days and compared with parallel 1G controls. After hypergravity treatment, whole flies and fly heads were separated, and evaluated for quantitative comparison of the two gravity conditions using an isobaric tagging liquid chromatography-tandem mass spectrometry approach. Data revealed a total of 1948 (whole flies) and 1480 (head) proteins to be differentially present in hypergravity-treated flies. Gene Ontology analysis of head specific proteomics revealed host immune response and humoral stress proteins were significantly upregulated. Proteins related to calcium signaling, ion transport and ATPase were decreased. Enhanced expression of cuticular proteins may suggest an alteration in chitin metabolism and in chitin-based cuticle development. We therefore present a comprehensive quantitative survey of proteomic changes in response to chronic hypergravity in Drosophila, which will help elucidate the underlying molecular mechanisms associated with altered gravity environments.

Hypergravity↗

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 NTRS1996

Identification of the cAMP response element that controls transcriptional activation of the insulin-like growth factor-I gene by prostaglandin E2 in osteoblasts

Insulin-like growth factor-I (IGF-I), a multifunctional growth factor, plays a key role in skeletal growth and can enhance bone cell replication and differentiation. We previously showed that prostaglandin E2 (PGE2) and other agents that increase cAMP activated IGF-I gene transcription in primary rat osteoblast cultures through promoter 1 (P1), the major IGF-I promoter, and found that transcriptional induction was mediated by protein kinase A. We now have identified a short segment of P1 that is essential for full hormonal regulation and have characterized inducible DNA-protein interactions involving this site. Transient transfections of IGF-I P1 reporter genes into primary rat osteoblasts showed that the 328-base pair untranslated region of exon 1 was required for a full 5.3-fold response to PGE2; mutation in a previously footprinted site, HS3D (base pairs +193 to +215), reduced induction by 65%. PGE2 stimulated nuclear protein binding to HS3D. Binding, as determined by gel mobility shift assay, was not seen in nuclear extracts from untreated osteoblast cultures, was detected within 2 h of PGE2 treatment, and was maximal by 4 h. This DNA-protein interaction was not observed in cytoplasmic extracts from PGE2-treated cultures, indicating nuclear localization of the protein kinase A-activated factor(s). Activation of this factor was not blocked by cycloheximide (Chx), and Chx did not impair stimulation of IGF-I gene expression by PGE2. In contrast, binding to a consensus cAMP response element (CRE; 5'-TGACGTCA-3') from the rat somatostatin gene was not modulated by PGE2 or Chx. Competition gel mobility shift analysis using mutated DNA probes identified 5'-CGCAATCG-3' as the minimal sequence needed for inducible binding. All modified IGF-I P1 promoterreporter genes with mutations within this CRE sequence also showed a diminished functional response to PGE2. These results identify the CRE within the 5'-untranslated region of IGF-I exon 1 that is required for hormonal activation of IGF-I gene transcription by cAMP in osteoblasts.

NASA Discipline Musculoskeletal↗

NASA NTRS2008

Closing the Phenotypic Gap between Transformed Neuronal Cell Lines in Culture and Untransformed Neurons

Studies of neuronal dysfunction in the central nervous system (CNS) are frequently limited by the failure of primary neurons to propagate in vitro. Neuronal cell lines can be substituted for primary cells but they often misrepresent normal conditions. We hypothesized that a dimensional (3-D) cell culture system would drive the phenotype of transformed neurons closer to that of untransformed cells. In our studies comparing 3-D versus 2-dimensional (2-D) culture, neuronal SH-SY5Y (SY) cells underwent distinct morphological changes combined with a significant drop in their rate of cell division. Expression of the proto-oncogene N-myc and the RNA binding protein HuD was decreased in 3-D culture as compared to standard 2-D conditions. We observed a decline in the anti-apoptotic protein Bcl-2 in 3-D culture, coupled with increased expression of the pro-apoptotic proteins Bax and Bak. Moreover, thapsigargin (TG)-induced apoptosis was enhanced in the 3-D cells. Microarray analysis demonstrated significantly differing mRNA levels for over 700 genes in the cells of each culture type. These results indicate that a 3-D culture approach narrows the phenotypic gap between neuronal cell lines and primary neurons. The resulting cells may readily be used for in vitro research of neuronal pathogenesis.

Myers, Tereance A.↗

NASA NTRS2000

An early ethylene up-regulated gene encoding a calmodulin-binding protein involved in plant senescence and death

35S-Labeled calmodulin (CaM) was used to screen a tobacco anther cDNA library. A positive clone (NtER1) with high homology to an early ethylene-up-regulated gene (ER66) in tomato, and an Arabidopsis homolog was isolated and characterized. Based on the helical wheel projection, a 25-mer peptide corresponding to the predicted CaM-binding region of NtER1 (amino acids 796-820) was synthesized. The gel-mobility shift assay showed that the peptide formed a stable complex with CaM only in the presence of Ca(2+). CaM binds to NtER1 with high affinity (K(d) approximately 12 nm) in a calcium-dependent manner. Tobacco flowers at different stages of development were treated with ethylene or with 1-methylcyclopropene for 2 h before treating with ethylene. Northern analysis showed that the NtER1 was rapidly induced after 15 min of exposure to ethylene. However, the 2-h 1-methylcyclopropene treatment totally blocked NtER1 expression in flowers at all stages of development, suggesting that NtER1 is an early ethylene-up-regulated gene. The senescing leaves and petals had significantly increased NtER1 induction as compared with young leaves and petals, implying that NtER1 is developmentally regulated and acts as a trigger for senescence and death. This is the first documented evidence for the involvement of Ca(2+)/CaM-mediated signaling in ethylene action.

Non-NASA Center↗

NASA NTRS2003

Genomic approaches to identifying transcriptional regulators of osteoblast differentiation

Recent microarray studies of mouse and human osteoblast differentiation in vitro have identified novel transcription factors that may be important in the establishment and maintenance of differentiation. These findings help unravel the pattern of gene-expression changes that underly the complex process of bone formation.

Non-NASA Center↗