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Results for “In Situ Hybridization”

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 55 records · Page 3
All sourcesNASA NTRSDOE OSTI
NASA NTRS1997

Dose reconstruction for individuals exposed to ionizing radiation using chromosome painting

To be most useful, a biomarker for dose reconstruction should employ an end point that is highly quantitative, stable with time and easily measured. Reciprocal translocations have been shown to be a promising biomarker that is linked to both prior exposure and risk, and they can be measured easily and quantitatively using fluorescence in situ hybridization. In contrast to other biomarkers that are available, the frequency of reciprocal translocations in individuals exposed to whole-body radiation is stable with time after exposure, has rather small interindividual variability and can be measured accurately at low levels of exposure. Results from recent studies demonstrate that measurements of reciprocal translocation frequencies, facilitated by chromosome painting, can be used to reconstruct radiation dose for individuals exposed in the distant past.

NASA Discipline Radiation Health↗
NASA NTRS1999

Developmental regulation of the gene for chimeric calcium/calmodulin-dependent protein kinase in anthers

Chimeric Ca(2+)/calmodulin-dependent protein kinase (CCaMK) was cloned from developing anthers of lily (Lilium longiflorum Thumb. cv. Nellie White) and tobacco (Nicotiana tabacum L. cv. Xanthi). Previous biochemical characterization and structure/function studies had revealed that CCaMK has dual modes of regulation by Ca(2+) and Ca(2+)/calmodulin. The unique structural features of CCaMK include a catalytic domain, a calmodulin-binding domain, and a neural visinin-like Ca(2+)-binding domain. The existence of these three features in a single polypeptide distinguishes it from other kinases. Western analysis revealed that CCaMK is expressed in a stage-specific manner in developing anthers. Expression of CCaMK was first detected in pollen mother cells and continued to increase, reaching a peak around the tetrad stage of meiosis. Following microsporogenesis, CCaMK expression rapidly decreased and at later stages of microspore development, no expression was detected. A tobacco genomic clone of CCaMK was isolated and transgenic tobacco plants were produced carrying the CCaMK promoter fused to the beta-glucuronidase reporter gene. Both CCaMK mRNA and protein were detected in the pollen sac and their localizations were restricted to the pollen mother cells and tapetal cells. Consistent results showing a stage-specific expression pattern were obtained by beta-glucuronidase analysis, in-situ hybridization and immunolocalization. The stage- and tissue-specific appearance of CCaMK in anthers suggests that it could play a role in sensing transient changes in free Ca(2+) concentration in target cells, thereby controlling developmental events in the anther.

NASA Discipline Plant Biology↗
NASA NTRS1996

Cloning and characterization of the murine homolog of the sno proto-oncogene reveals a novel splice variant

The cellular function(s) of the SNO protein remain undefined. To gain a better understanding of possible developmental roles of this cellular proto-oncogene, we have cloned two murine sno cDNAs and have investigated their expression patterns in embryonic and postnatal tissues. A single major transcript of 7.5 kb is detected in multiple tissues by Northern blot. However, reverse transcriptase polymerase chain reaction (RT-PCR) and RNAse protection assays revealed a novel splice variant in every tissue examined. Two isoforms, termed sno N and sno-dE3 (dE3, deletion within exon 3), were identified. The sno-dE3 isoform employs a novel 5' splice site located within the coding region of the third exon and deletes potential kinase recognition motifs. Transcripts of both sno isoforms accumulate ubiquitously but are most abundant in the developing central nervous system. The in situ hybridization patterns of sno expression during murine development suggest potential roles in tissues with a high degree of cellular proliferation. Expression in terminally differentiated tissues such as muscle and neurons indicates that SNO may have multiple functional activities.

Non-NASA Center↗
NASA NTRS1992

Identification of a mouse B-type cyclin which exhibits developmentally regulated expression in the germ line

To begin to examine the function of cyclins in mammalian germ cells, we have screened an adult mouse testis cDNA library for the presence of B-type cyclins. We have isolated cDNAs that encode a murine B-type cyclin, which has been designated cycB1. cycB1 was shown to be expressed in several adult tissues and in the midgestation mouse embryo. In the adult tissues, the highest levels of cycB1 transcripts were seen in the testis and ovary, which contain germ cells at various stages of differentiation. The major transcripts corresponding to cycB1 are 1.7 and 2.5 kb, with the 1.7 kb species being the predominant testicular transcript and the 2.5 kb species more abundant in the ovary. Examination of cDNAs corresponding to the 2.5 kb and 1.7 kb mRNAs revealed that these transcripts encode identical proteins, differing only in the polyadenylation signal used and therefore in the length of their 3' untranslated regions. Northern blot and in situ hybridization analyses revealed that the predominant sites of cycB1 expression in the testis and ovary were in the germinal compartment, particularly in early round spermatids in the testis and growing oocytes in the ovary. Thus cycB1 is expressed in both meiotic and postmeiotic cells. This pattern of cycB1 expression further suggests that cycB1 may have different functions in the two cell types, only one of which correlates with progression of the cell cycle.

NASA Discipline Developmental Biology↗
DOE OSTI2022

Robust, versatile DNA FISH probes for chromosome-specific repeats in Caenorhabditis elegans and Pristionchus pacificus

Repetitive DNA sequences are useful targets for chromosomal fluorescence in situ hybridization. We analyzed recent genome assemblies of Caenorhabditis elegans and Pristionchus pacificus to identify tandem repeats with a unique genomic localization. Based on these findings, we designed and validated sets of oligonucleotide probes for each species targeting at least 1 locus per chromosome. These probes yielded reliable fluorescent signals in different tissues and can easily be combined with the immunolocalization of cellular proteins. Synthesis and labeling of these probes are highly cost-effective and require no hands-on labor. The methods presented here can be easily applied in other model and nonmodel organisms with a sequenced genome.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1999

Estimate of the frequency of true incomplete exchanges in human lymphocytes exposed to 1 GeV/u Fe ions in vitro

PURPOSE: To study the frequency of true incomplete exchanges induced by high-LET radiation. MATERIALS AND METHODS: Human lymphocytes were exposed to 1 GeV/u Fe ions (LET = 140 keV/microm). Chromosome aberrations were analysed by a fluorescence in situ hybridization using a combination of whole-chromosome-specific probes and human telomere probes. Chromosomes 1, 3 and 4 were investigated. RESULTS: The percentage of incomplete exchanges was between 23 and 29% if telomere signals were not considered. The percentage decreased to approximately 10% after ruling out false incomplete exchanges containing telomere signals. The final estimation of true incomplete exchanges was <10%. CONCLUSION: Within a degree of uncertainty, the percentage of true incomplete exchanges in 1 GeV/u Fe ion-irradiated human lymphocytes was similar to that induced by gamma rays.

NASA Center JSC↗
NASA NTRS1997

Initiation of oncogenic transformation in human mammary epithelial cells by charged particles

Experimental studies have shown that high linear-energy transfer (LET) charged particles can be more effective than x-rays and gamma-rays in inducing oncogenic transformation in cultured cells and tumors in animals. Based on these results, experiments were designed and performed with an immortal human mammary epithelial cell line (H184B5), and several clones transformed by heavy ions were obtained. Cell fusion experiments were subsequently done, and results indicate that the transforming gene(s) is recessive. Chromosome analysis with fluorescence in situ hybridization (FISH) techniques also showed additional translocations in transformed human mammary epithelial cells. In addition, studies with these cell lines indicate that heavy ions can effectively induce deletion, break, and dicentrics. Deletion of tumor suppressor gene(s) and/or formation of translocation through DNA double strand breaks is a likely mechanism for the initiation of oncogenic transformation in human mammary epithelial cells.

NASA Discipline Radiation Health↗
NASA NTRS1997

Induction of chromosome aberrations in human cells by charged particles

Chromosome aberrations induced by high-energy charged particles in normal human lymphocytes and human fibroblasts have been investigated. The charged particles included 250 MeV/nucleon protons, 290 MeV/nucleon carbon ions and 1 GeV/nucleon iron ions. The energies of the charged particles were higher than in most of the studies reported in the literature. Lymphocytes were stimulated to grow immediately after irradiation, while fibroblasts were incubated at 37 degrees C for 24 h for repair. Chromosomes were collected at the first mitosis after irradiation and chromosome aberrations were scored using the fluorescence in situ hybridization (FISH) technique with a whole-chromosome 4 probe. Chromosome aberrations were classified as reciprocal exchanges, incomplete exchanges, deletions and complex exchanges. The relative biological effectiveness (RBE) for each type of aberration was calculated by dividing a dose of 4 Gy by the dose of the charged particles producing the same effect as 4 Gy of gamma rays. Results of this study showed that complex aberrations have the highest RBE for radiation of high linear energy transfer (LET) for human lymphocytes, but for fibroblasts, the greatest effect was for incomplete exchanges. For both lymphocytes and fibroblasts, iron ions induced a similar fraction of aberrant cells.

NASA Center JSC↗
NASA NTRS1996

Biological dosimetry by interphase chromosome painting

Both fluorescence in situ hybridization of metaphase spreads with whole-chromosome probes and premature chromosome condensation in interphase nuclei have been used in the past to estimate the radiation dose to lymphocytes. We combined these techniques to evaluate the feasibility of using painted interphase chromosomes for biodosimetry. Human peripheral lymphocytes were exposed to gamma rays and fused to mitotic Chinese hamster cells either immediately after irradiation or after 8 h incubation at 37 degrees C. Interphase or metaphase human chromosomes were hybridized with a composite probe specific for human chromosomes 3 and 4. The dose-response curve for fragment induction immediately after irradiation was linear; these results reflected breakage frequency in the total genome in terms of DNA content per chromosome. At 8 h after irradiation, the dose-response curve for chromosome interchanges, the prevalent aberration in interphase chromosomes, was linear-quadratic and similar to that observed for metaphase chromosomes. These results suggest that painting prematurely condensed chromosomes can be useful for biological dosimetry when blood samples are available shortly after the exposure, or when interphase cells are to be scored instead of mitotic cells.

NASA Discipline Radiation Health↗
NASA NTRS2002

Expression of the ctenophore Brain Factor 1 forkhead gene ortholog (ctenoBF-1) mRNA is restricted to the presumptive mouth and feeding apparatus: implications for axial organization in the Metazoa

Ctenophores are thoroughly modern animals whose ancestors are derived from a separate evolutionary branch than that of other eumetazoans. Their major longitudinal body axis is the oral-aboral axis. An apical sense organ, called the apical organ, is located at the aboral pole and contains a highly innervated statocyst and photodetecting cells. The apical organ integrates sensory information and controls the locomotory apparatus of ctenophores, the eight longitudinal rows of ctene/comb plates. In an effort to understand the developmental and evolutionary organization of axial properties of ctenophores we have isolated a forkhead gene from the Brain Factor 1 (BF-1) family. This gene, ctenoBF-1, is the first full-length nuclear gene reported from ctenophores. This makes ctenophores the most basal metazoan (to date) known to express definitive forkhead class transcription factors. Orthologs of BF-1 in vertebrates, Drosophila, and Caenorhabditis elegans are expressed in anterior neural structures. Surprisingly, in situ hybridizations with ctenoBF-1 antisense riboprobes show that this gene is not expressed in the apical organ of ctenophores. CtenoBF-1 is expressed prior to first cleavage. Transcripts become localized to the aboral pole by the 8-cell stage and are inherited by ectodermal micromeres generated from this region at the 16- and 32-cell stages. Expression in subsets of these cells persists and is seen around the edge of the blastopore (presumptive mouth) and in distinct ectodermal regions along the tentacular poles. Following gastrulation, stomodeal expression begins to fade and intense staining becomes restricted to two distinct domains in each tentacular feeding apparatus. We suggest that the apical organ is not homologous to the brain of bilaterians but that the oral pole of ctenophores corresponds to the anterior pole of bilaterian animals.

Non-NASA Center↗
DOE OSTI2021

LncRNA LINC00680 promotes lung adenocarcinoma growth via binding to GATA6 and canceling GATA6-mediated suppression of SOX12 expression

Lung adenocarcinoma (LUAD) is a major subtype of non-small-cell lung cancers (NSCLC). LINC00680 has been characterized as a novel oncogenic lncRNA in LUAD, but its regulatory mechanisms remain largely unclear. This study aimed to explore the subcellular localization of LINC00680 in LUAD and its regulation on the transcriptional process. LUAD cell lines (A549, H1650, and H1299) were used for in vitro and in vivo studies. Results showed LINC00680 depletion resulted in G0/G1 phase arrest of LUAD cells and reduced CDK4 and cyclin D1 expression in H1650 and H1299 cells. LINC00680 overexpression promoted A549 cell proliferation and increased CDK4 and cyclin D1 expression. RNA-fluorescence in situ hybridization (FISH) assay showed that LINC00680 has both cytoplasmic and nuclear distribution in LUAD cells. RNA pulldown and western blotting assays confirmed a physical interaction between LINC00680 and GATA6. In LUAD cells, GATA6 overexpression only slightly suppressed SOX12 transcription. ChIP-qPCR and dual-luciferase assay showed that GATA6 only weakly bound to the SOX12 promoter and decreased its activity. However, when LINC00680 was depleted, these transcriptional suppressive effects were significantly enhanced. These findings suggested that LINC00680 forms a complex with GATA6 and weakens its transcriptional suppressive effect on SOX12 expression. In the nude mice model, LINC00680 overexpression partly abrogated the growth-suppressive effects of GATA6 on A549 derived tumors. In summary, this study revealed a novel LINC00680-GATA6-SOX12 axis in promoting LUAD cell cycle progression and proliferation. Future studies should be conducted for a better understanding of the complex networking of LINC00680 in LUAD.

60 APPLIED LIFE SCIENCES↗
NASA NTRS2001

Twisting integrin receptors increases endothelin-1 gene expression in endothelial cells

A magnetic twisting stimulator was developed based on the previously published technique of magnetic twisting cytometry. Using ligand-coated ferromagnetic microbeads, this device can apply mechanical stresses with varying amplitudes, duration, frequencies, and waveforms to specific cell surface receptors. Biochemical and biological responses of the cells to the mechanical stimulation can be assayed. Twisting integrin receptors with RGD (Arg-Gly-Asp)-containing peptide-coated beads increased endothelin-1 (ET-1) gene expression by >100%. In contrast, twisting scavenger receptors with acetylated low-density lipoprotein-coated beads or twisting HLA antigen with anti-HLA antibody-coated beads did not lead to alterations in ET-1 gene expression. In situ hybridization showed that the increase in ET-1 mRNA was localized in the cells that were stressed with the RGD-coated beads. Blocking stretch-activated ion channels with gadolinium, chelating Ca2+ with EGTA, or inhibiting tyrosine phosphorylation with genistein abolished twist-induced ET-1 mRNA elevation. Abolishing cytoskeletal tension with an inhibitor of the myosin ATPase, with an inhibitor of myosin light chain kinase, or with an actin microfilament disrupter blocked twisted-induced increases in ET-1 expression. Our results are consistent with the hypothesis that the molecular structural linkage of integrin-cytoskeleton is an important pathway for stress-induced ET-1 gene expression.

Non-NASA Center↗
NASA NTRS1997

Biodosimetry results from space flight Mir-18

Astronauts are classified as radiation workers due to the presence of ionizing radiation in space. For the assessment of health risks, physical dosimetry has been indispensable. However, the change of the location of dosimeters on the crew members, the variation in dose rate with location inside the spacecraft and the unknown biological effects of microgravity can introduce significant uncertainties in estimating exposure. To circumvent such uncertainty, a study on the cytogenetic effects of space radiation in human lymphocytes was proposed and conducted for Mir-18, a 115-day mission. This study used fluorescence in situ hybridization (FISH) with whole-chromosome painting probes to score chromosomal exchanges and the Giemsa staining method to determine the frequency of dicentrics. The growth kinetics of cells and sister chromatid exchanges (SCEs) were examined to ensure that chromosomal aberrations were scored in the first mitosis and were induced primarily by space radiation. Our results showed that the frequency of chromosomal aberrations increased significantly in postflight samples compared to samples drawn prior to flight, and that the frequency of SCEs was similar for both pre- and postflight samples. Based on a dose-response curve for preflight samples exposed to gamma rays, the absorbed dose received by crew members during the mission was estimated to be about 14.75 cSv. Because the absorbed dose measured by physical dosimeters is 5.2 cGy for the entire mission, the RBE is about 2.8.

NASA Discipline Radiation Health↗
NASA NTRS2003

Biological Effectiveness of Accelerated Particles for the Induction of Chromosome Damage Measured in Metaphase and Interphase Human Lymphocytes

Chromosome aberrations were investigated in human lymphocytes after in vitro exposure to 1H-, 3He-, 12C-, 40Ar-, 28Si-, 56Fe-, or 197Au-ion beams, with LET ranging from approximately 0.4-1393 keV/microm in the dose range of 0.075-3 Gy. Dose-response curves for chromosome exchanges, measured at the first mitosis postirradiation using fluorescence in situ hybridization (FISH) with whole-chromosome probes, were fitted with linear or linear-quadratic functions. The relative biological effectiveness (RBE) was estimated from the initial slope of the dose-response curve for chromosomal damage with respect to low- or high-dose-rate gamma rays. Estimates of RBEmax values for mitotic spreads, which ranged from near 0.7 to 11.1 for total exchanges, increased with LET, reaching a maximum at about 150 keV/microm, and decreased with further increase in LET. RBEs for complex aberrations are undefined due to the lack of an initial slope for gamma rays. Additionally, the effect of mitotic delay on RBE values was investigated by measuring chromosome aberrations in interphase after chemically induced premature chromosome condensation (PCC), and values were up to threefold higher than for metaphase analysis.

NASA Discipline Radiation Health↗
NASA NTRS1999

Theoretical and experimental tests of a chromosomal fingerprint for densely ionizing radiation based on F ratios calculated from stable and unstable chromosome aberrations

In the present study, F ratios for both stable chromosome aberrations, i.e. ratios of translocations to pericentric inversions, and unstable aberrations, i.e. dicentrics and centric rings, were measured using fluorescence in situ hybridization. F ratios for stable aberrations measured after exposure to low (2.89 Gy 60Co gamma rays) and high-LET (0.25 Gy 56Fe ions; 1.25 Gy 56Fe ions; 3.0 Gy 12C ions) radiation were 6.5 +/- 1.5, 4.7 +/- 1.6, 9.3 +/- 2.5 and 10.4 +/- 3.0, respectively. F ratios for unstable aberrations measured after low (2.89 Gy 60Co gamma rays) and high-LET (0.25 Gy 56Fe ions; 3.0 Gy 12C ions) radiations were 6.5 +/- 1.6, 6.3 +/- 2.3 and 11.1 +/- 3.7, respectively. No significant difference between the F ratios for low- and high-LET radiation was found. Further tests on the models for calculation of the F ratio proposed by Brenner and Sachs (Radiat. Res. 140, 134-142, 1994) showed that the F ratio may not be straightforward as a practical fingerprint for densely ionizing radiation.

NASA Center JSC↗
NASA NTRS1996

Rejoining and misrejoining of radiation-induced chromatin breaks. II. Biophysical Model

A biophysical model for the kinetics of the formation of radiation-induced chromosome aberrations is developed to account for the recent experimental results obtained with a combination of the premature chromosome condensation (PCC) and fluorescence in situ hybridization (FISH) techniques. In this model, we consider the broken ends of DNA double-strand breaks (DSBs) to be reactant and make use of the interaction distance hypothesis. The repair/misrepair process between broken ends is suggested to consist of two steps; the first step represents the two break ends approaching each other, and the second step represents the enzymatic processes leading to DNA end-to-end rejoining. Only the second step is reflected in the kinetics observed in experiments using PCC. The model appears to be able to fit existing data for human cells. It is shown that the kinetics of the formation of chromosome aberrations can be explained by a single rate that characterizes both rejoining and misrejoining of DSBs, suggesting that repair and misrepair share the same mechanism. Fast repair (completed in minutes) in a subset of DSBs is suggested as an explanation of the complete exchanges observed with PCC in human lymphocytes immediately after irradiation. The fast repair component seems to be absent in human fibroblasts.

NASA Center JSC↗
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 NTRS1999

Critical target and dose and dose-rate responses for the induction of chromosomal instability by ionizing radiation

To investigate the critical target, dose response and dose-rate response for the induction of chromosomal instability by ionizing radiation, bromodeoxyuridine (BrdU)-substituted and unsubstituted GM10115 cells were exposed to a range of doses (0.1-10 Gy) and different dose rates (0.092-17.45 Gy min(-1)). The status of chromosomal stability was determined by fluorescence in situ hybridization approximately 20 generations after irradiation in clonal populations derived from single progenitor cells surviving acute exposure. Overall, nearly 700 individual clones representing over 140,000 metaphases were analyzed. In cells unsubstituted with BrdU, a dose response was found, where the probability of observing delayed chromosomal instability in any given clone was 3% per gray of X rays. For cells substituted with 25-66% BrdU, however, a dose response was observed only at low doses (<1.0 Gy); at higher doses (>1.0 Gy), the incidence of chromosomal instability leveled off. There was an increase in the frequency and complexity of chromosomal instability per unit dose compared to cells unsubstituted with BrdU. The frequency of chromosomal instability appeared to saturate around approximately 30%, an effect which occurred at much lower doses in the presence of BrdU. Changing the gamma-ray dose rate by a factor of 190 (0.092 to 17.45 Gy min(-1)) produced no significant differences in the frequency of chromosomal instability. The enhancement of chromosomal instability promoted by the presence of the BrdU argues that DNA comprises at least one of the critical targets important for the induction of this end point of genomic instability.

NASA Discipline Radiation Health↗