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Results for “RNA, Messenger/genetics”

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.

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At least 19 records
All sourcesNASA NTRSDOE OSTI
NASA NTRS1992

Regulation of an H-ras-related transcript by parathyroid hormone in rat osteosarcoma cells

The rat osteosarcoma cell line UMR 106-01 is a commonly used model system for the study of osteoblast function. However, it also expresses a phenotype characteristic of transformed cells. To test whether the latter could be accounted for by aberrant oncogene expression, we probed Northern blots of UMR and other osteoblastic cells with a panel of oncogene probes. These blots, when probed with a cDNA specific for v-H-ras, revealed a 7.0-kilobase (kb) H-ras-related transcript (designated HRRT) in UMR 106-01 cells that was not expressed in other osteoblastic cells. Osteoblast-enriched calvarial cells expressed the typical 1.1-kb H-ras mRNA, which was absent in UMR cells. Additionally, Western blots of lysates of UMR cells documented the presence of three proteins immunologically related to H-rasp21. To determine whether HRRT represented a recombinant retrovirus product, Northern blots were probed with a cDNA specific for the highly conserved gag-pol region of Moloney murine leukemia virus. These blots showed parallel cross-reactivity with an apparently identical transcript of 7.0 kb. The 7.0-kb transcripts detected by both v-H-ras and gag-pol probes declined to the same extent after treatment with concentrations of PTH known to inhibit proliferation of these cells. PTH regulated the abundance of HRRT in a time- and dose-dependent manner, with greatest repression of the transcript after 8 h of treatment with 10(-8) M PTH. The decrease in HRRT could not be completely accounted for by changes in transcriptional activity, as determined by nuclear run-on assays.(ABSTRACT TRUNCATED AT 250 WORDS).

Non-NASA Center↗
NASA NTRS2001

Light differentially regulates cell division and the mRNA abundance of pea nucleolin during de-etiolation

The abundance of plant nucleolin mRNA is regulated during de-etiolation by phytochrome. A close correlation between the mRNA abundance of nucleolin and mitosis has also been previously reported. These results raised the question of whether the effects of light on nucleolin mRNA expression were a consequence of light effects on mitosis. To test this we compared the kinetics of light-mediated increases in cell proliferation with that of light-mediated changes in the abundance of nucleolin mRNA using plumules of dark-grown pea (Pisum sativum) seedlings. These experiments show that S-phase increases 9 h after a red light pulse, followed by M-phase increases in the plumule leaves at 12 h post-irradiation, a time course consistent with separately measured kinetics of red light-induced increases in the expression of cell cycle-regulated genes. These increases in cell cycle-regulated genes are photoreversible, implying that the light-induced increases in cell proliferation are, like nucleolin mRNA expression, regulated via phytochrome. Red light stimulates increases in the mRNA for nucleolin at 6 h post-irradiation, prior to any cell proliferation changes and concurrent with the reported timing of phytochrome-mediated increases of rRNA abundance. After a green light pulse, nucleolin mRNA levels increase without increasing S-phase or M-phase. Studies in animals and yeast indicate that nucleolin plays a significant role in ribosome biosynthesis. Consistent with this function, pea nucleolin can rescue nucleolin deletion mutants of yeast that are defective in rRNA synthesis. Our data show that during de-etiolation, the increased expression of nucleolin mRNA is more directly regulated by light than by mitosis.

NASA Program Fundamental Space Biology↗
NASA NTRS2001

Structure of the coding region and mRNA variants of the apyrase gene from pea (Pisum sativum)

Partial amino acid sequences of a 49 kDa apyrase (ATP diphosphohydrolase, EC 3.6.1.5) from the cytoskeletal fraction of etiolated pea stems were used to derive oligonucleotide DNA primers to generate a cDNA fragment of pea apyrase mRNA by RT-PCR and these primers were used to screen a pea stem cDNA library. Two almost identical cDNAs differing in just 6 nucleotides within the coding regions were found, and these cDNA sequences were used to clone genomic fragments by PCR. Two nearly identical gene fragments containing 8 exons and 7 introns were obtained. One of them (H-type) encoded the mRNA sequence described by Hsieh et al. (1996) (DDBJ/EMBL/GenBank Z32743), while the other (S-type) differed by the same 6 nucleotides as the mRNAs, suggesting that these genes may be alleles. The six nucleotide differences between these two alleles were found solely in the first exon, and these mutation sites had two types of consensus sequences. These mRNAs were found with varying lengths of 3' untranslated regions (3'-UTR). There are some similarities between the 3'-UTR of these mRNAs and those of actin and actin binding proteins in plants. The putative roles of the 3'-UTR and alternative polyadenylation sites are discussed in relation to their possible role in targeting the mRNAs to different subcellular compartments.

NASA Discipline Plant Biology↗
NASA NTRS1996

Expression of spicule matrix protein gene SM30 in embryonic and adult mineralized tissues of sea urchin Hemicentrotus pulcherrimus

We have isolated a cDNA clone for spicule matrix protein, SM30, from sea urchin Hemicentrotus pulcherrimus and have studied the expression of this gene in comparison with that of another spicule matrix protein gene, SM50. In cultured micromeres as well as in intact embryos transcripts of SM30 were first detectable around the onset of spicule formation and rapidly increased with the growth of spicules, which accompanied accumulation of glycosylated SM30 protein(s). When micromeres were cultured in the presence of Zn2+, spicule formation and SM30 expression were suppressed, while both events resumed concurrently after the removal of Zn2+ from the culture medium. Expression of SM50, in contrast, started before the appearance of spicules and was not sensitive to Zn2+. Differences were also observed in adult tissues; SM30 mRNA was detected in spines and tube feet but not in the test, while SM50 mRNA was apparent in all of these mineralized tissues at similar levels. These results strongly suggest that the SM30 gene is regulated by a different mechanism to that of the SM50 gene and that the products of these two genes are differently involved in sea urchin biomineralization. A possible role of SM30 protein in skeleton formation is discussed.

NASA Discipline Musculoskeletal↗
NASA NTRS1996

Regulation of contractile protein gene expression in unloaded mouse skeletal muscle

Hindlimb unloading was performed on mice in an effort to study the regulation of contractile protein genes. In particular, the regulation of myosin heavy chain IIb was examined. During unloading, muscle fibers undergo a type conversion. Preliminary data from this study does not support the hypothesis that the fiber type conversion is due to an increase in promoter activity of fast isoform genes, such as myosin heavy chain IIb. The consequences of this finding are examined, with particular focus on other factors controlling gene regulation.

Non-NASA Center↗
NASA NTRS1992

Gene activity during germination of spores of the fern, Onoclea sensibilis. Cell-free translation analysis of mRNA of spores and the effect of alpha-amanitin on spore germination

Poly(A)-RNA fractions of dormant, dark-imbibed (non-germinating) and photoinduced (germinating) spores of Onoclea sensibilis were poor templates in the rabbit reticulocyte lysate protein synthesizing system, but the translational efficiency of poly(A)+RNA was considerably higher than that of unfractionated RNA. Poly(A)+RNA isolated from photoinduced spores had a consistently higher translational efficiency than poly(A)+RNA from dark-imbibed spores. Analysis of the translation products by one-dimensional polyacrylamide gel electrophoresis showed no qualitative differences in the mRNA populations of dormant, dark-imbibed, and photoinduced spores. However, poly(A)+RNA from dark-imbibed spores appeared to encode in vitro fewer detectable polypeptides at a reduced intensity than photoinduced spores. A DNA clone encoding the large subunit of maize ribulose bisphosphate carboxylase hybridized at strong to moderate intensity to RNA isolated from dark-imbibed spores, indicating the absence of mRNA degradation. Although alpha-amanitin did not inhibit the germination of spores, the drug prevented the elongation of the rhizoid and protonemal initial with a concomitant effect on the synthesis of poly(A)+RNA. These results are consistent with the view that some form of translational control involving stored mRNA operates during dark-imbibition and photoinduced germination of spores.

NASA Discipline Number 29-20↗
NASA NTRS1991

Gene activity during germination of spores of the fern, Onoclea sensibilis: RNA and protein synthesis and the role of stored mRNA

Pattern of 3H-uridine incorporation into RNA of spores of Onoclea sensibilis imbibed in complete darkness (non-germinating conditions) and induced to germinate in red light was followed by oligo-dT cellulose chromatography, gel electrophoresis coupled with fluorography and autoradiography. In dark-imbibed spores, RNA synthesis was initiated about 24 h after sowing, with most of the label accumulating in the high mol. wt. poly(A) -RNA fraction. There was no incorporation of the label into poly(A) +RNA until 48 h after sowing. In contrast, photo-induced spores began to synthesize all fractions of RNA within 12 h after sowing and by 24 h, incorporation of 3H-uridine into RNA of irradiated spores was nearly 70-fold higher than that into dark-imbibed spores. Protein synthesis, as monitored by 3H-arginine incorporation into the acid-insoluble fraction and by autoradiography, was initiated in spores within 1-2 h after sowing under both conditions. Autoradiographic experiments also showed that onset of protein synthesis in the cytoplasm of the germinating spore is independent of the transport of newly synthesized nuclear RNA. One-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis of 35S-methionine-labelled proteins revealed a good correspondence between proteins synthesized in a cell-free translation system directed by poly(A) +RNA of dormant spores and those synthesized in vivo by dark-imbibed and photo-induced spores. These results indicate that stored mRNAs of O. sensibilis spores are functionally competent and provide templates for the synthesis of proteins during dark-imbibition and germination.

NASA Discipline Number 29-20↗
NASA NTRS2003

Inhibition of HSP70 and a Collagen-Specific Molecular Chaperone (HSP47) Expression in Rat Osteoblasts by Microgravity

Rat osteoblasts were cultured aboard a space shuttle for 4 or 5 days. Cells were exposed to 1alpha, 25 dihydroxyvitamin D(3) during the last 20 h and then solubilized by guanidine solution. The mRNA levels for molecular chaperones were analyzed by semi-quantitative RT-PCR. ELISA was used to quantify TGF-beta1 in the conditioned medium. The HSP70 mRNA levels in the flight cultures were almost completely suppressed, as compared to the ground (1 x g) controls. The inducible HSP70 is known as the major heat shock protein that prevents stress-induced apoptosis. The mean mRNA levels for the constitutive HSC73 in the flight cultures were reduced to 69%, approximately 60% of the ground controls. HSC73 is reported to prevent the pathological state that is induced by disruption of microtubule network. The mean HSP47 mRNA levels in the flight cultures were decreased to 50% and 19% of the ground controls on the 4th and 5th days. Concomitantly, the concentration of TGF-beta1 in the conditioned medium of the flight cultures was reduced to 37% and 19% of the ground controls on the 4th and 5th days. HSP47 is the collagen-specific molecular chaperone that controls collagen processing and quality and is regulated by TGF-beta1. Microgravity differentially modulated the expression of molecular chaperones in osteoblasts, which might be involved in induction and/or prevention of osteopenia in space.

Animals↗
NASA NTRS2001

Dirigent proteins and dirigent sites in lignifying tissues

Tissue-specific dirigent protein gene expression and associated dirigent (site) localization were examined in various organs of Forsythia intermedia using tissue printing, in situ mRNA hybridization and immunolabeling techniques, respectively. Dirigent protein gene expression was primarily noted in the undifferentiated cambial regions of stem sections, whereas dirigent protein sites were detected mainly in the vascular cambium and ray parenchyma cell initials. Immunolocalization also revealed cross-reactivity with particular regions of the lignified cell walls, these being coincident with the known sites of initiation of lignin deposition. These latter regions are considered to harbor contiguous arrays of dirigent (monomer binding) sites for initiation of lignin biopolymer assembly. Dirigent protein mRNA expression was also localized in the vascular regions of roots and petioles, whereas in leaves the dirigent sites were primarily associated with the palisade layers and the vascular bundle. That is, dirigent protein mediated lignan biosynthesis was initiated primarily in the cambium and ray cell initial regions of stems as well as in the leaf palisade layers, this being in accordance with the occurrence of the lignans for defense purposes. Within lignified secondary xylem cell walls, however, dirigent sites were primarily localized in the S(1) sublayer and compound middle lamella, these being coincident with previously established sites for initiation of macromolecular lignin biosynthesis. Once initiation occurs, lignification is proposed to continue through template polymerization.

NASA Discipline Plant Biology↗
NASA NTRS2000

Selected contribution: a three-dimensional model for assessment of in vitro toxicity in balaena mysticetus renal tissue

This study established two- and three-dimensional renal proximal tubular cell cultures of the endangered species bowhead whale (Balaena mysticetus), developed SV40-transfected cultures, and cloned the 61-amino acid open reading frame for the metallothionein protein, the primary binding site for heavy metal contamination in mammals. Microgravity research, modulations in mechanical culture conditions (modeled microgravity), and shear stress have spawned innovative approaches to understanding the dynamics of cellular interactions, gene expression, and differentiation in several cellular systems. These investigations have led to the creation of ex vivo tissue models capable of serving as physiological research analogs for three-dimensional cellular interactions. These models are enabling studies in immune function, tissue modeling for basic research, and neoplasia. Three-dimensional cellular models emulate aspects of in vivo cellular architecture and physiology and may facilitate environmental toxicological studies aimed at elucidating biological functions and responses at the cellular level. Marine mammals occupy a significant ecological niche (72% of the Earth's surface is water) in terms of the potential for information on bioaccumulation and transport of terrestrial and marine environmental toxins in high-order vertebrates. Few ex vivo models of marine mammal physiology exist in vitro to accomplish the aforementioned studies. Techniques developed in this investigation, based on previous tissue modeling successes, may serve to facilitate similar research in other marine mammals.

Whales/genetics/physiology↗
NASA NTRS2001

Fatty acid regulates gene expression and growth of human prostate cancer PC-3 cells

It has been proposed that the omega-6 fatty acids increase the rate of tumor growth. Here we test that hypothesis in the PC-3 human prostate tumor. We found that the essential fatty acids, linoleic acid (LA) and arachidonic acid (AA), and the AA metabolite PGE(2) stimulate tumor growth while oleic acid (OA) and the omega-3 fatty acid, eicosapentaenoic acid (EPA) inhibited growth. In examining the role of AA in growth response, we extended our studies to analyze changes in early gene expression induced by AA. We demonstrate that c-fos expression is increased within minutes of addition in a dose-dependent manner. Moreover, the immediate early gene cox-2 is also increased in the presence of AA in a dose-dependent manner, while the constitutive cox-1 message was not increased. Three hours after exposure to AA, the synthesis of PGE(2) via COX-2 was also increased. Previous studies have demonstrated that AA was primarily delivered by low density lipoprotein (LDL) via its receptor (LDLr). Since it is known that hepatomas, acute myelogenous leukemia and colorectal tumors lack normal cholesterol feedback, we examined the role of the LDLr in growth regulation of the PC-3 prostate cancer cells. Analysis of ldlr mRNA expression and LDLr function demonstrated that human PC-3 prostate cancer cells lack normal feedback regulation. While exogenous LDL caused a significant stimulation of cell growth and PGE(2) synthesis, no change was seen in regulation of the LDLr by LDL. Taken together, these data show that normal cholesterol feedback of ldlr message and protein is lost in prostate cancer. These data suggest that unregulated over-expression of LDLr in tumor cells would permit increased availability of AA, which induces immediate early genes c-fos and cox-2 within minutes of uptake.

NASA Discipline Cell Biology↗
NASA NTRS2000

Developmental regulation of collagenase-3 mRNA in normal, differentiating osteoblasts through the activator protein-1 and the runt domain binding sites

Collagenase-3 mRNA is initially detectable when osteoblasts cease proliferation, increasing during differentiation and mineralization. We showed that this developmental expression is due to an increase in collagenase-3 gene transcription. Mutation of either the activator protein-1 or the runt domain binding site decreased collagenase-3 promoter activity, demonstrating that these sites are responsible for collagenase-3 gene transcription. The activator protein-1 and runt domain binding sites bind members of the activator protein-1 and core-binding factor family of transcription factors, respectively. We identified core-binding factor a1 binding to the runt domain binding site and JunD in addition to a Fos-related antigen binding to the activator protein-1 site. Overexpression of both c-Fos and c-Jun in osteoblasts or core-binding factor a1 increased collagenase-3 promoter activity. Furthermore, overexpression of c-Fos, c-Jun, and core-binding factor a1 synergistically increased collagenase-3 promoter activity. Mutation of either the activator protein-1 or the runt domain binding site resulted in the inability of c-Fos and c-Jun or core-binding factor a1 to increase collagenase-3 promoter activity, suggesting that there is cooperative interaction between the sites and the proteins. Overexpression of Fra-2 and JunD repressed core-binding factor a1-induced collagenase-3 promoter activity. Our results suggest that members of the activator protein-1 and core-binding factor families, binding to the activator protein-1 and runt domain binding sites are responsible for the developmental regulation of collagenase-3 gene expression in osteoblasts.

NASA Discipline Musculoskeletal↗
NASA NTRS1999

Ubiquitin conjugation by the N-end rule pathway and mRNAs for its components increase in muscles of diabetic rats

Insulin deficiency (e.g., in acute diabetes or fasting) is associated with enhanced protein breakdown in skeletal muscle leading to muscle wasting. Because recent studies have suggested that this increased proteolysis is due to activation of the ubiquitin-proteasome (Ub-proteasome) pathway, we investigated whether diabetes is associated with an increased rate of Ub conjugation to muscle protein. Muscle extracts from streptozotocin-induced insulin-deficient rats contained greater amounts of Ub-conjugated proteins than extracts from control animals and also 40-50% greater rates of conjugation of (125)I-Ub to endogenous muscle proteins. This enhanced Ub-conjugation occurred mainly through the N-end rule pathway that involves E2(14k) and E3alpha. A specific substrate of this pathway, alpha-lactalbumin, was ubiquitinated faster in the diabetic extracts, and a dominant negative form of E2(14k) inhibited this increase in ubiquitination rates. Both E2(14k) and E3alpha were shown to be rate-limiting for Ub conjugation because adding small amounts of either to extracts stimulated Ub conjugation. Furthermore, mRNA for E2(14k) and E3alpha (but not E1) were elevated 2-fold in muscles from diabetic rats, although no significant increase in E2(14k) and E3alpha content could be detected by immunoblot or activity assays. The simplest interpretation of these results is that small increases in both E2(14k) and E3alpha in muscles of insulin-deficient animals together accelerate Ub conjugation and protein degradation by the N-end rule pathway, the same pathway activated in cancer cachexia, sepsis, and hyperthyroidism.

Non-NASA Center↗
NASA NTRS1998

BIGEL analysis of gene expression in HL60 cells exposed to X rays or 60 Hz magnetic fields

We screened a panel of 1,920 randomly selected cDNAs to discover genes that are differentially expressed in HL60 cells exposed to 60 Hz magnetic fields (2 mT) or X rays (5 Gy) compared to unexposed cells. Identification of these clones was accomplished using our two-gel cDNA library screening method (BIGEL). Eighteen cDNAs differentially expressed in X-irradiated compared to control HL60 cells were recovered from a panel of 1,920 clones. Differential expression in experimental compared to control cells was confirmed independently by Northern blotting of paired total RNA samples hybridized to each of the 18 clone-specific cDNA probes. DNA sequencing revealed that 15 of the 18 cDNA clones produced matches with the database for genes related to cell growth, protein synthesis, energy metabolism, oxidative stress or apoptosis (including MYC, neuroleukin, copper zinc-dependent superoxide dismutase, TC4 RAS-like protein, peptide elongation factor 1alpha, BNIP3, GATA3, NF45, cytochrome c oxidase II and triosephosphate isomerase mRNAs). In contrast, BIGEL analysis of the same 1,920 cDNAs revealed no differences greater than 1.5-fold in expression levels in magnetic-field compared to sham-exposed cells. Magnetic-field-exposed and control samples were analyzed further for the presence of mRNA encoding X-ray-responsive genes by hybridization of the 18 specific cDNA probes to RNA from exposed and control HL60 cells. Our results suggest that differential gene expression is induced in approximately 1% of a random pool of cDNAs by ionizing radiation but not by 60 Hz magnetic fields under the present experimental conditions.

Non-NASA Center↗
NASA NTRS1994

The beetle Tribolium castaneum has a fushi tarazu homolog expressed in stripes during segmentation

The genetic control of embryonic organization is far better understood for the fruit fly Drosophila melanogaster than for any other metazoan. A gene hierarchy acts during oogenesis and embryogenesis to regulate the establishment of segmentation along the anterior-posterior axis, and homeotic selector genes define developmental commitments within each parasegmental unit delineated. One of the most intensively studied Drosophila segmentation genes is fushi tarazu (ftz), a pair-rule gene expressed in stripes that is important for the establishment of the parasegmental boundaries. Although ftz is flanked by homeotic selector genes conserved throughout the metazoa, there is no evidence that it was part of the ancestral homeotic complex, and it has been unclear when the gene arose and acquired a role in segmentation. We show here that the beetle Tribolium castaneum has a ftz homolog located in its Homeotic complex and expressed in a pair-rule fashion, albeit in a register differing from that of the fly gene. These and other observations demonstrate that a ftz gene preexisted the radiation of holometabolous insects and suggest that it has a role in beetle embryogenesis which differs somewhat from that described in flies.

NASA Discipline Developmental Biology↗
NASA NTRS1993

Evolution of EF-hand calcium-modulated proteins. III. Exon sequences confirm most dendrograms based on protein sequences: calmodulin dendrograms show significant lack of parallelism

In the first report in this series we presented dendrograms based on 152 individual proteins of the EF-hand family. In the second we used sequences from 228 proteins, containing 835 domains, and showed that eight of the 29 subfamilies are congruent and that the EF-hand domains of the remaining 21 subfamilies have diverse evolutionary histories. In this study we have computed dendrograms within and among the EF-hand subfamilies using the encoding DNA sequences. In most instances the dendrograms based on protein and on DNA sequences are very similar. Significant differences between protein and DNA trees for calmodulin remain unexplained. In our fourth report we evaluate the sequences and the distribution of introns within the EF-hand family and conclude that exon shuffling did not play a significant role in its evolution.

Non-NASA Center↗
NASA NTRS2004

Modeled microgravity-induced protein kinase C isoform expression in human lymphocytes

In long-term space travel, the crew is exposed to microgravity and radiation that invoke potential hazards to the immune system. T cell activation is a critical step in the immune response. Receptor-mediated signaling is inhibited in both microgravity and modeled microgravity (MMG) as reflected by diminished DNA synthesis in peripheral blood lymphocytes and their locomotion through gelled type I collagen. Direct activation of protein kinase C (PKC) bypassing cell surface events using the phorbol ester PMA rescues MMG-inhibited lymphocyte activation and locomotion, whereas the calcium ionophore ionomycin had no rescue effect. Thus calcium-independent PKC isoforms may be affected in MMG-induced locomotion inhibition and rescue. Both calcium-dependent isoforms and calcium-independent PKC isoforms were investigated to assess their expression in lymphocytes in 1 g and MMG culture. Human lymphocytes were cultured and harvested at 24, 48, 72, and 96 h, and serial samples were assessed for locomotion by using type I collagen and expression of PKC isoforms. Expression of PKC-alpha, -delta, and -epsilon was assessed by RT-PCR, flow cytometry, and immunoblotting. Results indicated that PKC isoforms delta and epsilon were downregulated by >50% at the transcriptional and translational levels in MMG-cultured lymphocytes compared with 1-g controls. Events upstream of PKC, such as phosphorylation of phospholipase Cgamma in MMG, revealed accumulation of inactive enzyme. Depressed calcium-independent PKC isoforms may be a consequence of an upstream lesion in the signal transduction pathway. The differential response among calcium-dependent and calcium-independent isoforms may actually result from MMG intrusion events earlier than PKC, but after ligand-receptor interaction.

Lymphocytes/enzymology↗