Molecular Analysis and Genomic Organization of Major DNA Satellites in Banana (Musa spp.)

PubMed Central

Čížková, Jana; Hřibová, Eva; Humplíková, Lenka; Christelová, Pavla; Suchánková, Pavla; Doležel, Jaroslav

2013-01-01

Satellite DNA sequences consist of tandemly arranged repetitive units up to thousands nucleotides long in head-to-tail orientation. The evolutionary processes by which satellites arise and evolve include unequal crossing over, gene conversion, transposition and extra chromosomal circular DNA formation. Large blocks of satellite DNA are often observed in heterochromatic regions of chromosomes and are a typical component of centromeric and telomeric regions. Satellite-rich loci may show specific banding patterns and facilitate chromosome identification and analysis of structural chromosome changes. Unlike many other genomes, nuclear genomes of banana (Musa spp.) are poor in satellite DNA and the information on this class of DNA remains limited. The banana cultivars are seed sterile clones originating mostly from natural intra-specific crosses within M. acuminata (A genome) and inter-specific crosses between M. acuminata and M. balbisiana (B genome). Previous studies revealed the closely related nature of the A and B genomes, including similarities in repetitive DNA. In this study we focused on two main banana DNA satellites, which were previously identified in silico. Their genomic organization and molecular diversity was analyzed in a set of nineteen Musa accessions, including representatives of A, B and S (M. schizocarpa) genomes and their inter-specific hybrids. The two DNA satellites showed a high level of sequence conservation within, and a high homology between Musa species. FISH with probes for the satellite DNA sequences, rRNA genes and a single-copy BAC clone 2G17 resulted in characteristic chromosome banding patterns in M. acuminata and M. balbisiana which may aid in determining genomic constitution in interspecific hybrids. In addition to improving the knowledge on Musa satellite DNA, our study increases the number of cytogenetic markers and the number of individual chromosomes, which can be identified in Musa. PMID:23372772

Molecular analysis and genomic organization of major DNA satellites in banana (Musa spp.).

PubMed

Čížková, Jana; Hřibová, Eva; Humplíková, Lenka; Christelová, Pavla; Suchánková, Pavla; Doležel, Jaroslav

2013-01-01

Satellite DNA sequences consist of tandemly arranged repetitive units up to thousands nucleotides long in head-to-tail orientation. The evolutionary processes by which satellites arise and evolve include unequal crossing over, gene conversion, transposition and extra chromosomal circular DNA formation. Large blocks of satellite DNA are often observed in heterochromatic regions of chromosomes and are a typical component of centromeric and telomeric regions. Satellite-rich loci may show specific banding patterns and facilitate chromosome identification and analysis of structural chromosome changes. Unlike many other genomes, nuclear genomes of banana (Musa spp.) are poor in satellite DNA and the information on this class of DNA remains limited. The banana cultivars are seed sterile clones originating mostly from natural intra-specific crosses within M. acuminata (A genome) and inter-specific crosses between M. acuminata and M. balbisiana (B genome). Previous studies revealed the closely related nature of the A and B genomes, including similarities in repetitive DNA. In this study we focused on two main banana DNA satellites, which were previously identified in silico. Their genomic organization and molecular diversity was analyzed in a set of nineteen Musa accessions, including representatives of A, B and S (M. schizocarpa) genomes and their inter-specific hybrids. The two DNA satellites showed a high level of sequence conservation within, and a high homology between Musa species. FISH with probes for the satellite DNA sequences, rRNA genes and a single-copy BAC clone 2G17 resulted in characteristic chromosome banding patterns in M. acuminata and M. balbisiana which may aid in determining genomic constitution in interspecific hybrids. In addition to improving the knowledge on Musa satellite DNA, our study increases the number of cytogenetic markers and the number of individual chromosomes, which can be identified in Musa.

High-throughput analysis of the satellitome illuminates satellite DNA evolution

NASA Astrophysics Data System (ADS)

Ruiz-Ruano, Francisco J.; López-León, María Dolores; Cabrero, Josefa; Camacho, Juan Pedro M.

2016-07-01

Satellite DNA (satDNA) is a major component yet the great unknown of eukaryote genomes and clearly underrepresented in genome sequencing projects. Here we show the high-throughput analysis of satellite DNA content in the migratory locust by means of the bioinformatic analysis of Illumina reads with the RepeatExplorer and RepeatMasker programs. This unveiled 62 satDNA families and we propose the term “satellitome” for the whole collection of different satDNA families in a genome. The finding that satDNAs were present in many contigs of the migratory locust draft genome indicates that they show many genomic locations invisible by fluorescent in situ hybridization (FISH). The cytological pattern of five satellites showing common descent (belonging to the SF3 superfamily) suggests that non-clustered satDNAs can become into clustered through local amplification at any of the many genomic loci resulting from previous dissemination of short satDNA arrays. The fact that all kinds of satDNA (micro- mini- and satellites) can show the non-clustered and clustered states suggests that all these elements are mostly similar, except for repeat length. Finally, the presence of VNTRs in bacteria, showing similar properties to non-clustered satDNAs in eukaryotes, suggests that this kind of tandem repeats show common properties in all living beings.

Interaction of the alpha-subunit of Escherichia coli RNA polymerase with DNA: rigid body nature of the protein-DNA contact.

PubMed

Heyduk, E; Baichoo, N; Heyduk, T

2001-11-30

The alpha-subunit of Escherichia coli RNA polymerase plays an important role in the activity of many promoters by providing a direct protein-DNA contact with a specific sequence (UP element) located upstream of the core promoter sequence. To obtain insight into the nature of thermodynamic forces involved in the formation of this protein-DNA contact, the binding of the alpha-subunit of E. coli RNA polymerase to a fluorochrome-labeled DNA fragment containing the rrnB P1 promoter UP element sequence was quantitatively studied using fluorescence polarization. The alpha dimer and DNA formed a 1:1 complex in solution. Complex formation at 25 degrees C was enthalpy-driven, the binding was accompanied by a net release of 1-2 ions, and no significant specific ion effects were observed. The van't Hoff plot of temperature dependence of binding was linear suggesting that the heat capacity change (Deltac(p)) was close to zero. Protein footprinting with hydroxyradicals showed that the protein did not change its conformation upon protein-DNA contact formation. No conformational changes in the DNA molecule were detected by CD spectroscopy upon protein-DNA complex formation. The thermodynamic characteristics of the binding together with the lack of significant conformational changes in the protein and in the DNA suggested that the alpha-subunit formed a rigid body-like contact with the DNA in which a tight complementary recognition interface between alpha-subunit and DNA was not formed.

Satellite DNA methylation status and expression of selected genes in Bos indicus blastocysts produced in vivo and in vitro.

PubMed

Urrego, R; Bernal-Ulloa, S M; Chavarría, N A; Herrera-Puerta, E; Lucas-Hahn, A; Herrmann, D; Winkler, S; Pache, D; Niemann, H; Rodriguez-Osorio, N

2017-04-01

Bovine embryos produced in vivo and in vitro differ with respect to molecular profiles, including epigenetic marks and gene expression profiles. This study investigated the CpG methylation status in bovine testis satellite I (BTS) and Bos taurus alpha satellite I (BTαS) DNA sequences, and concomitantly the relative abundance of transcripts, critically involved in DNA methylation (DNMT1 and DNMT3A), growth and development (IGF2R) and pluripotency (POU5F1) in Bos indicus embryos produced in vitro or in vivo. Results revealed that methylation of BTS were higher (P < 0.05) in embryos produced in vitro compared with their in vivo produced counterparts, while the methylation status of BTαS was similar in both groups. There were no significant differences in transcript abundance for DNMT3A, IGF2R and POU5F1 between blastocysts produced in vivo and in vitro. However, a significantly lower amount of DNMT1 transcripts was found in the in vitro cultured embryos (P < 0.05) compared with their in vivo derived counterparts. In conclusion, this study reported only minor changes in the expression of developmentally important genes and satellite DNA methylation related to the in vitro embryo production system.

Isolation of human hexosaminidase. cap alpha. cDNA and expression of. cap alpha. chains in E. coli

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wiktorowicz, J.E.; Whitman, J.M.

1986-05-01

Pooled antisera against homogeneous, glutaraldehyde cross-linked hexosaminidase (hex) A was adsorbed with E. coli lysate insolubilized on Sepharose 4B. Aliquots of a human liver lambdagtll cDNA library (50,000-100,000 pfu) were plated on E. coli Y1090. Expression of cloned cDNA, after sufficient plaque growth at 42/sup 0/, was accomplished by induction with isopropylthiogalactoside soaked nitrocellulose filters. Identification of hex cDNA clones was performed by incubation of the filters with purified antisera. Protein A labelled with I-125 was used to develop the reactive plaques. Positive plaques, identified by autoradiography, were picked, replated at a lower density, and rescreened. This was repeated severalmore » more times until all plaques yielded positive signals. Identification of the clones as containing ..cap alpha.. or ..beta.. cDNA was accomplished by replating the purified phage and rescreening the plaques with anti-hex B antiserum preadsorbed with E. coli lysate. According to this protocol several hex ..cap alpha.. clones have been identified. While these clones generate ..beta..-galactosidase: hex ..cap alpha.. fusion proteins, these findings suggest that in the future it may be possible to obtain large quantities of unmodified hex ..cap alpha.. and ..beta.. polypeptides from E. coli for the study of the structural and enzymatic properties of these polypeptides and for diagnostic purposes in the GM2 gangliosidoses.« less

Separation of detector non-linearity issues and multiple ionization satellites in alpha-particle PIXE

NASA Astrophysics Data System (ADS)

Campbell, John L.; Ganly, Brianna; Heirwegh, Christopher M.; Maxwell, John A.

2018-01-01

Multiple ionization satellites are prominent features in X-ray spectra induced by MeV energy alpha particles. It follows that the accuracy of PIXE analysis using alpha particles can be improved if these features are explicitly incorporated in the peak model description when fitting the spectra with GUPIX or other codes for least-squares fitting PIXE spectra and extracting element concentrations. A method for this incorporation is described and is tested using spectra recorded on Mars by the Curiosity rover's alpha particle X-ray spectrometer. These spectra are induced by both PIXE and X-ray fluorescence, resulting in a spectral energy range from ∼1 to ∼25 keV. This range is valuable in determining the energy-channel calibration, which departs from linearity at low X-ray energies. It makes it possible to separate the effects of the satellites from an instrumental non-linearity component. The quality of least-squares spectrum fits is significantly improved, raising the level of confidence in analytical results from alpha-induced PIXE.

A novel nanometric DNA thin film as a sensor for alpha radiation

PubMed Central

Kulkarni, Atul; Kim, Byeonghoon; Dugasani, Sreekantha Reddy; Joshirao, Pranav; Kim, Jang Ah; Vyas, Chirag; Manchanda, Vijay; Kim, Taesung; Park, Sung Ha

2013-01-01

The unexpected nuclear accidents have provided a challenge for scientists and engineers to develop sensitive detectors, especially for alpha radiation. Due to the high linear energy transfer value, sensors designed to detect such radiation require placement in close proximity to the radiation source. Here we report the morphological changes and optical responses of artificially designed DNA thin films in response to exposure to alpha radiation as observed by an atomic force microscope, a Raman and a reflectance spectroscopes. In addition, we discuss the feasibility of a DNA thin film as a radiation sensing material. The effect of alpha radiation exposure on the DNA thin film was evaluated as a function of distance from an 241Am source and exposure time. Significant reflected intensity changes of the exposed DNA thin film suggest that a thin film made of biomolecules can be one of promising candidates for the development of online radiation sensors. PMID:23792924

Association of pKi-67 with satellite DNA of the human genome in early G1 cells.

PubMed

Bridger, J M; Kill, I R; Lichter, P

1998-01-01

pKi-67 is a nucleolar antigen that provides a specific marker for proliferating cells. It has been shown previously that pKi-67's distribution varies in a cell cycle-dependent manner: it coats all chromosomes during mitosis, accumulates in nuclear foci during G1 phase (type I distribution) and localizes within nucleoli in late G1 S and G2 phase (type II distribution). Although no function has as yet been ascribed to pKi-67, it has been found associated with centromeres in G1. In the present study the distribution pattern of pKi-67 during G1 in human dermal fibroblasts (HDFs) was analysed in more detail. Synchronization experiments show that in very early G1 cells pKi-67 coincides with virtually all satellite regions analysed, i.e. with centromeric (alpha-satellite), telomeric (minisatellite) and heterochromatic blocks (satellite III) on chromosomes 1 and Y (type Ia distribution). In contrast, later in the G1 phase, a smaller fraction of satellite DNA regions are found collocalized with pKi-67 foci (type Ib distribution). When all pKi-67 becomes localized within nucleoli, even fewer satellite regions remain associated with the pKi-67 staining. However, all centromeric and short arm regions of the acrocentric chromosomes, which are in very close proximity to or even contain the rRNA genes, are collocalized with anti-pKi-67 staining throughout the remaining interphase of the cell cycle. Thus, our data demonstrate that during post-mitotic reformation and nucleogenesis there is a progressive decline in the fraction of specific satellite regions of DNA that remain associated with pKi-67. This may be relevant to nucleolar reformation following mitosis.

α satellite DNA variation and function of the human centromere

PubMed Central

Sullivan, Lori L.; Chew, Kimberline

2017-01-01

ABSTRACT Genomic variation is a source of functional diversity that is typically studied in genic and non-coding regulatory regions. However, the extent of variation within noncoding portions of the human genome, particularly highly repetitive regions, and the functional consequences are not well understood. Satellite DNA, including α satellite DNA found at human centromeres, comprises up to 10% of the genome, but is difficult to study because its repetitive nature hinders contiguous sequence assemblies. We recently described variation within α satellite DNA that affects centromere function. On human chromosome 17 (HSA17), we showed that size and sequence polymorphisms within primary array D17Z1 are associated with chromosome aneuploidy and defective centromere architecture. However, HSA17 can counteract this instability by assembling the centromere at a second, “backup” array lacking variation. Here, we discuss our findings in a broader context of human centromere assembly, and highlight areas of future study to uncover links between genomic and epigenetic features of human centromeres. PMID:28406740

DNA condensation by chiral alpha-methylated polyamine analogues and protection of cellular DNA from oxidative damage.

PubMed

Nayvelt, Irina; Hyvönen, Mervi T; Alhonen, Leena; Pandya, Ipsit; Thomas, Thresia; Khomutov, Alex R; Vepsäläinen, Jouko; Patel, Rajesh; Keinänen, Tuomo A; Thomas, T J

2010-01-11

Polyamines are essential molecules supporting the structure, conformation, and function of nucleic acids and proteins. We studied stereoisomers of alpha,alpha'-dimethylated spermine [(R,R)-Me(2)Spm, (S,S)-Me(2)Spm, (R,S)-Me(2)Spm] for their ability to provoke DNA condensation and protect DNA from damage. (R,R)- and (R,S)-Me(2)Spm displayed more efficient condensing ability than spermine, with significantly lower EC(50) (concentration for 50% compaction) values (p < or = 0.01). However, spermine exerted slightly more duplex stabilization than Me(2)Spm. Condensation resulted in nanoparticles with hydrodynamic radii between 39.6 and 48.4 nm, and electron microscopy showed the presence of toroids and spheroids. Natural polyamines and stereoisomers of Me(2)Spm protected DNA against DNase digestion and oxidative stress in vitro and against etoposide and oxidative stress in DU145 cells but afforded little protection against UV-C irradiation. Our findings indicate that Me(2)Spm stereoisomers are efficient DNA packaging agents with potential applications in gene delivery. Our study also reveals stereospecificity in DNA interaction and protection against cellular stress.

Satellite DNA Sequences in Canidae and Their Chromosome Distribution in Dog and Red Fox.

PubMed

Vozdova, Miluse; Kubickova, Svatava; Cernohorska, Halina; Fröhlich, Jan; Rubes, Jiri

2016-01-01

Satellite DNA is a characteristic component of mammalian centromeric heterochromatin, and a comparative analysis of its evolutionary dynamics can be used for phylogenetic studies. We analysed satellite and satellite-like DNA sequences available in NCBI for 4 species of the family Canidae (red fox, Vulpes vulpes, VVU; domestic dog, Canis familiaris, CFA; arctic fox, Vulpes lagopus, VLA; raccoon dog, Nyctereutes procyonoides procyonoides, NPR) by comparative sequence analysis, which revealed 86-90% intraspecies and 76-79% interspecies similarity. Comparative fluorescence in situ hybridisation in the red fox and dog showed signals of the red fox satellite probe in canine and vulpine autosomal centromeres, on VVUY, B chromosomes, and in the distal parts of VVU9q and VVU10p which were shown to contain nucleolus organiser regions. The CFA satellite probe stained autosomal centromeres only in the dog. The CFA satellite-like DNA did not show any significant sequence similarity with the satellite DNA of any species analysed and was localised to the centromeres of 9 canine chromosome pairs. No significant heterochromatin block was detected on the B chromosomes of the red fox. Our results show extensive heterogeneity of satellite sequences among Canidae and prove close evolutionary relationships between the red and arctic fox. © 2017 S. Karger AG, Basel.

Organization and evolution of highly repeated satellite DNA sequences in plant chromosomes.

PubMed

Sharma, S; Raina, S N

2005-01-01

A major component of the plant nuclear genome is constituted by different classes of repetitive DNA sequences. The structural, functional and evolutionary aspects of the satellite repetitive DNA families, and their organization in the chromosomes is reviewed. The tandem satellite DNA sequences exhibit characteristic chromosomal locations, usually at subtelomeric and centromeric regions. The repetitive DNA family(ies) may be widely distributed in a taxonomic family or a genus, or may be specific for a species, genome or even a chromosome. They may acquire large-scale variations in their sequence and copy number over an evolutionary time-scale. These features have formed the basis of extensive utilization of repetitive sequences for taxonomic and phylogenetic studies. Hybrid polyploids have especially proven to be excellent models for studying the evolution of repetitive DNA sequences. Recent studies explicitly show that some repetitive DNA families localized at the telomeres and centromeres have acquired important structural and functional significance. The repetitive elements are under different evolutionary constraints as compared to the genes. Satellite DNA families are thought to arise de novo as a consequence of molecular mechanisms such as unequal crossing over, rolling circle amplification, replication slippage and mutation that constitute "molecular drive". Copyright 2005 S. Karger AG, Basel.

DNA methylation at a bovine alpha satellite I repeat CpG site during development following fertilization and somatic cell nuclear transfer.

PubMed

Couldrey, Christine; Wells, David N

2013-01-01

Incomplete epigenetic reprogramming is postulated to contribute to the low developmental success following somatic cell nuclear transfer (SCNT). Here, we describe the epigenetic reprogramming of DNA methylation at an alpha satellite I CpG site (αsatI-5) during development of cattle generated either by artificial insemination (AI) or in vitro fertilization (IVF) and SCNT. Quantitative methylation analysis identified that SCNT donor cells were highly methylated at αsatI-5 and resulting SCNT blastocysts showed significantly more methylation than IVF blastocysts. At implantation, no difference in methylation was observed between SCNT and AI in trophoblast tissue at αsatI-5, however, SCNT embryos were significantly hyper-methylated compared to AI controls at this time point. Following implantation, DNA methylation at αsatI-5 decreased in AI but not SCNT placental tissues. In contrast to placenta, the proportion of methylation at αsatI-5 remained high in adrenal, kidney and muscle tissues during development. Differences in the average proportion of methylation were smaller in somatic tissues than placental tissues but, on average, SCNT somatic tissues were hyper-methylated at αsatI-5. Although sperm from all bulls was less methylated than somatic tissues at αsatI-5, on average this site remained hyper-methylated in sperm from cloned bulls compared with control bulls. This developmental time course confirms that epigenetic reprogramming does occur, at least to some extent, following SCNT. However, the elevated methylation levels observed in SCNT blastocysts and cellular derivatives implies that there is either insufficient time or abundance of appropriate reprogramming factors in oocytes to ensure complete reprogramming. Incomplete reprogramming at this CpG site may be a contributing factor to low SCNT success rates, but more likely represents the tip of the iceberg in terms of incompletely reprogramming. Until protocols ensure the epigenetic signature of a

DNA Methylation at a Bovine Alpha Satellite I Repeat CpG Site during Development following Fertilization and Somatic Cell Nuclear Transfer

PubMed Central

Couldrey, Christine; Wells, David N.

2013-01-01

Incomplete epigenetic reprogramming is postulated to contribute to the low developmental success following somatic cell nuclear transfer (SCNT). Here, we describe the epigenetic reprogramming of DNA methylation at an alpha satellite I CpG site (αsatI-5) during development of cattle generated either by artificial insemination (AI) or in vitro fertilization (IVF) and SCNT. Quantitative methylation analysis identified that SCNT donor cells were highly methylated at αsatI-5 and resulting SCNT blastocysts showed significantly more methylation than IVF blastocysts. At implantation, no difference in methylation was observed between SCNT and AI in trophoblast tissue at αsatI-5, however, SCNT embryos were significantly hyper-methylated compared to AI controls at this time point. Following implantation, DNA methylation at αsatI-5 decreased in AI but not SCNT placental tissues. In contrast to placenta, the proportion of methylation at αsatI-5 remained high in adrenal, kidney and muscle tissues during development. Differences in the average proportion of methylation were smaller in somatic tissues than placental tissues but, on average, SCNT somatic tissues were hyper-methylated at αsatI-5. Although sperm from all bulls was less methylated than somatic tissues at αsatI-5, on average this site remained hyper-methylated in sperm from cloned bulls compared with control bulls. This developmental time course confirms that epigenetic reprogramming does occur, at least to some extent, following SCNT. However, the elevated methylation levels observed in SCNT blastocysts and cellular derivatives implies that there is either insufficient time or abundance of appropriate reprogramming factors in oocytes to ensure complete reprogramming. Incomplete reprogramming at this CpG site may be a contributing factor to low SCNT success rates, but more likely represents the tip of the iceberg in terms of incompletely reprogramming. Until protocols ensure the epigenetic signature of a

Detection of alpha particles using DNA/Al Schottky junctions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Al-Ta'ii, Hassan Maktuff Jaber, E-mail: hassankirkukly@gmail.com, E-mail: vengadeshp@um.edu.my; Department of Physics, Faculty of Science, University of Al-Muthana, Al-Muthana 66001; Periasamy, Vengadesh, E-mail: hassankirkukly@gmail.com, E-mail: vengadeshp@um.edu.my

2015-09-21

Deoxyribonucleic acid or DNA can be utilized in an organic-metallic rectifying structure to detect radiation, especially alpha particles. This has become much more important in recent years due to crucial environmental detection needs in both peace and war. In this work, we fabricated an aluminum (Al)/DNA/Al structure and generated current–voltage characteristics upon exposure to alpha radiation. Two models were utilized to investigate these current profiles; the standard conventional thermionic emission model and Cheung and Cheung's method. Using these models, the barrier height, Richardson constant, ideality factor and series resistance of the metal-DNA-metal structure were analyzed in real time. The barriermore » height, Φ value calculated using the conventional method for non-radiated structure was 0.7149 eV, increasing to 0.7367 eV after 4 min of radiation. Barrier height values were observed to increase after 20, 30 and 40 min of radiation, except for 6, 8, and 10 min, which registered a decrease of about 0.67 eV. This was in comparison using Cheung and Cheung's method, which registered 0.6983 eV and 0.7528 eV for the non-radiated and 2 min of radiation, respectively. The barrier height values, meanwhile, were observed to decrease after 4 (0.61 eV) to 40 min (0.6945 eV). The study shows that conventional thermionic emission model could be practically utilized for estimating the diode parameters including the effect of series resistance. These changes in the electronic properties of the Al/DNA/Al junctions could therefore be utilized in the manufacture of sensitive alpha particle sensors.« less

The structures of non-CG-repeat Z-DNAs co-crystallized with the Z-DNA-binding domain, hZ alpha(ADAR1).

PubMed

Ha, Sung Chul; Choi, Jongkeun; Hwang, Hye-Yeon; Rich, Alexander; Kim, Yang-Gyun; Kim, Kyeong Kyu

2009-02-01

The Z-DNA conformation preferentially occurs at alternating purine-pyrimidine repeats, and is specifically recognized by Z alpha domains identified in several Z-DNA-binding proteins. The binding of Z alpha to foreign or chromosomal DNA in various sequence contexts is known to influence various biological functions, including the DNA-mediated innate immune response and transcriptional modulation of gene expression. For these reasons, understanding its binding mode and the conformational diversity of Z alpha bound Z-DNAs is of considerable importance. However, structural studies of Z alpha bound Z-DNA have been mostly limited to standard CG-repeat DNAs. Here, we have solved the crystal structures of three representative non-CG repeat DNAs, d(CACGTG)(2), d(CGTACG)(2) and d(CGGCCG)(2) complexed to hZ alpha(ADAR1) and compared those structures with that of hZ alpha(ADAR1)/d(CGCGCG)(2) and the Z alpha-free Z-DNAs. hZ alpha(ADAR1) bound to each of the three Z-DNAs showed a well conserved binding mode with very limited structural deviation irrespective of the DNA sequence, although varying numbers of residues were in contact with Z-DNA. Z-DNAs display less structural alterations in the Z alpha-bound state than in their free form, thereby suggesting that conformational diversities of Z-DNAs are restrained by the binding pocket of Z alpha. These data suggest that Z-DNAs are recognized by Z alpha through common conformational features regardless of the sequence and structural alterations.

Satellite DNA Modulates Gene Expression in the Beetle Tribolium castaneum after Heat Stress

PubMed Central

Feliciello, Isidoro; Akrap, Ivana; Ugarković, Đurđica

2015-01-01

Non-coding repetitive DNAs have been proposed to perform a gene regulatory role, however for tandemly repeated satellite DNA no such role was defined until now. Here we provide the first evidence for a role of satellite DNA in the modulation of gene expression under specific environmental conditions. The major satellite DNA TCAST1 in the beetle Tribolium castaneum is preferentially located within pericentromeric heterochromatin but is also dispersed as single repeats or short arrays in the vicinity of protein-coding genes within euchromatin. Our results show enhanced suppression of activity of TCAST1-associated genes and slower recovery of their activity after long-term heat stress relative to the same genes without associated TCAST1 satellite DNA elements. The level of gene suppression is not influenced by the distance of TCAST1 elements from the associated genes up to 40 kb from the genes’ transcription start sites, but it does depend on the copy number of TCAST1 repeats within an element, being stronger for the higher number of copies. The enhanced gene suppression correlates with the enrichment of the repressive histone marks H3K9me2/3 at dispersed TCAST1 elements and their flanking regions as well as with increased expression of TCAST1 satellite DNA. The results reveal transient, RNAi based heterochromatin formation at dispersed TCAST1 repeats and their proximal regions as a mechanism responsible for enhanced silencing of TCAST1-associated genes. Differences in the pattern of distribution of TCAST1 elements contribute to gene expression diversity among T. castaneum strains after long-term heat stress and might have an impact on adaptation to different environmental conditions. PMID:26275223

Satellite DNA and cytogenetic evolution: molecular aspects and implications for man. [Kangaroo rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hatch, F.T.; Mazrimas, J.

1977-02-28

Simple, highly reiterated DNA sequences, often observed in density gradients as satellite DNAs, exist in condensed heterochromatin. This material is predominantly located at chromosomal centromeres, occasionally at telomeres, or intercalated within arms; in a few species it occupies entire chromosome arms. Satellite DNAs are a highly variable component of the genome of most higher eukaryotes, but their functions have remained speculative. The genus of kangaroo rats (Dipodomys) exhibits remarkable interspecies variations in content of three satellite DNAs, consisting of simple sequences 3 to 10 base pairs long, and in species karyotypes. A broad range of diploid-DNA content is correlated withmore » satellite-DNA content. The latter is correlated positively with predominance of biarmed over uniarmed chromosomes (high fundamental number FN) and inversely with two anatomical indices (leg-bone-length ratios) of specialization for the jumping gait. Karyotypic variation is achieved via chromosomal rearrangements, e.g., Robertsonian fusion, C-band heteromorphism, and pericentric inversion. Environmental adaptation is achieved, in part, by reassortment of gene-linkage groups and regulatory controls as a result of the chromosomal rearrangements. The foregoing relationships led to the postulation that highly reiterated DNA sequences play a supragenic, global role in environmental adaptation and the evolution of new species.« less

5-bp Classical Satellite DNA Loci from Chromosome-1 Instability in Cervical Neoplasia Detected by DNA Breakage Detection/Fluorescence in Situ Hybridization (DBD-FISH).

PubMed

Cortés-Gutiérrez, Elva I; Ortíz-Hernández, Brenda L; Dávila-Rodríguez, Martha I; Cerda-Flores, Ricardo M; Fernández, José Luis; López-Fernández, Carmen; Gosálvez, Jaime

2013-02-19

We aimed to evaluate the association between the progressive stages of cervical neoplasia and DNA damage in 5-bp classical satellite DNA sequences from chromosome-1 in cervical epithelium and in peripheral blood lymphocytes using DNA breakage detection/fluorescence in situ hybridization (DBD-FISH). A hospital-based unmatched case-control study was conducted in 2011 with a sample of 30 women grouped according to disease stage and selected according to histological diagnosis; 10 with low-grade squamous intraepithelial lesions (LG-SIL), 10 with high-grade SIL (HG-SIL), and 10 with no cervical lesions, from the Unidad Medica de Alta Especialidad of The Mexican Social Security Institute, IMSS, Mexico. Specific chromosome damage levels in 5-bp classical satellite DNA sequences from chromosome-1 were evaluated in cervical epithelium and peripheral blood lymphocytes using the DBD-FISH technique. Whole-genome DNA hybridization was used as a reference for the level of damage. Results of Kruskal-Wallis test showed a significant increase according to neoplastic development in both tissues. The instability of 5-bp classical satellite DNA sequences from chromosome-1 was evidenced using chromosome-orientation FISH. In conclusion, we suggest that the progression to malignant transformation involves an increase in the instability of 5-bp classical satellite DNA sequences from chromosome-1.

Satellite DNA and Transposable Elements in Seabuckthorn (Hippophae rhamnoides), a Dioecious Plant with Small Y and Large X Chromosomes

PubMed Central

Puterova, Janka; Razumova, Olga; Martinek, Tomas; Alexandrov, Oleg; Divashuk, Mikhail; Kubat, Zdenek; Hobza, Roman; Karlov, Gennady

2017-01-01

Seabuckthorn (Hippophae rhamnoides) is a dioecious shrub commonly used in the pharmaceutical, cosmetic, and environmental industry as a source of oil, minerals and vitamins. In this study, we analyzed the transposable elements and satellites in its genome. We carried out Illumina DNA sequencing and reconstructed the main repetitive DNA sequences. For data analysis, we developed a new bioinformatics approach for advanced satellite DNA analysis and showed that about 25% of the genome consists of satellite DNA and about 24% is formed of transposable elements, dominated by Ty3/Gypsy and Ty1/Copia LTR retrotransposons. FISH mapping revealed X chromosome-accumulated, Y chromosome-specific or both sex chromosomes-accumulated satellites but most satellites were found on autosomes. Transposable elements were located mostly in the subtelomeres of all chromosomes. The 5S rDNA and 45S rDNA were localized on one autosomal locus each. Although we demonstrated the small size of the Y chromosome of the seabuckthorn and accumulated satellite DNA there, we were unable to estimate the age and extent of the Y chromosome degeneration. Analysis of dioecious relatives such as Shepherdia would shed more light on the evolution of these sex chromosomes. PMID:28057732

High resolution DNA melting analysis: an application for prenatal control of alpha-thalassemia.

PubMed

Sirichotiyakul, Supatra; Wanapirak, Chanane; Saetung, Rattika; Sanguansermsri, Torpong

2010-04-01

To report the use of real-time gap-PCR using SYTO9 with high-resolution melting analysis (HRMA) in prenatal diagnosis of alpha-thalassemia 1. Real-time gap-PCR using SYTO9 with HRMA was performed in 33 DNA samples from chorionic villi sampling (8 normal, 16 heterozygous, and 9 homozygous) to determine the alpha-thalassemia 1 gene [normal and Southeast Asia (-SEA) allele]. The dissociation curve analysis in normal and - SEA allele gave a peak of T(m) at 91.80 +/- 0.14 degrees C and 88.67 +/- 0.08 degrees C, respectively. Normal genotype and homozygous alpha-thalassemia 1 showed a single peak of T(m) that corresponded to their alleles. The heterozygotes gave both peaks with higher normal peak and smaller - SEA peak. Thirty one samples showed consistent results with the conventional gap-PCR. Two samples with ambiguous results were confirmed to be maternal DNA contamination on real-time quantitative PCR and microsatellite assay. HRMA from both samples showed similar pattern to that of heterozygotes. However, they showed much smaller normal peak compared with the - SEA peak, which is in contrast to those of heterozygotes and can readily be distinguished. HRMA with SYTO9 is feasible for prenatal diagnosis of alpha-thalassemia. It had potential advantage of prompt detection maternal DNA contamination. Copyright (c) 2010 John Wiley & Sons, Ltd.

Construction of a small Mus musculus repetitive DNA library: identification of a new satellite sequence in Mus musculus.

PubMed Central

Pietras, D F; Bennett, K L; Siracusa, L D; Woodworth-Gutai, M; Chapman, V M; Gross, K W; Kane-Haas, C; Hastie, N D

1983-01-01

We report the construction of a small library of recombinant plasmids containing Mus musculus repetitive DNA inserts. The repetitive cloned fraction was derived from denatured genomic DNA by reassociation to a Cot value at which repetitive, but not unique, sequences have reannealed followed by exhaustive S1 nuclease treatment to degrade single stranded DNA. Initial characterizations of this library by colony filter hybridizations have led to the identification of a previously undetected M. musculus minor satellite as well as to clones containing M. musculus major satellite sequences. This new satellite is repeated 10-20 times less than the major satellite in the M. musculus genome. It has a repeat length of 130 nucleotides compared with the M. musculus major satellite with a repeat length of 234 nucleotides. Sequence analysis of the minor satellite has shown that it has a 29 base pair region with extensive homology to one of the major satellite repeating subunits. We also show by in situ hybridization that this minor satellite sequence is located at the centromeres and possibly the arms of at least half the M musculus chromosomes. Sequences related to the minor satellite have been found in the DNA of a related Mus species, Mus spretus, and may represent the major satellite of that species. Images PMID:6314268

cap alpha. /sub i/-3 cDNA encodes the. cap alpha. subunit of G/sub k/, the stimulatory G protein of receptor-regulated K/sup +/ channels

DOE Office of Scientific and Technical Information (OSTI.GOV)

Codina, J.; Olate, J.; Abramowitz, J.

1988-05-15

cDNA cloning has identified the presence in the human genome of three genes encoding ..cap alpha.. subunits of pertussis toxin substrates, generically called G/sub i/. They are named ..cap alpha../sub i/-1, ..cap alpha../sub i/-2 and ..cap alpha../sub i/-3. However, none of these genes has been functionally identified with any of the ..cap alpha.. subunits of several possible G proteins, including pertussis toxin-sensitive G/sub p/'s, stimulatory to phospholipase C or A/sub 2/, G/sub i/, inhibitory to adenylyl cyclase, or G/sub k/, stimulatory to a type of K/sup +/ channels. The authors now report the nucleotide sequence and the complete predicted aminomore » acid sequence of human liver ..cap alpha../sub i/-3 and the partial amino acid sequence of proteolytic fragments of the ..cap alpha.. subunit of human erythrocyte G/sub k/. The amino acid sequence of the proteolytic fragment is uniquely encoded by the cDNA of ..cap alpha../sub i/-3, thus identifying it as ..cap alpha../sub k/. The probable identity of ..cap alpha../sub i/-1 with ..cap alpha../sub p/ and possible roles for ..cap alpha../sub i/-2, as well as additional roles for ..cap alpha../sub i/-1 and ..cap alpha../sub i/-3 (..cap alpha../sub k/) are discussed.« less

Effect of alpha-ketoglutarate and oxaloacetate on brain mitochondrial DNA damage and seizures induced by kainic acid in mice.

PubMed

Yamamoto, Hiro-aki; Mohanan, Parayanthala V

2003-07-20

The effects of alpha-ketoglutarate and oxaloacetate on brain mitochondrial DNA (mtDNA) damage and seizures induced by kainic acid were examined both in vivo and in vitro. An intraperitoneal (ip) injection of kainic acid (45 mg/kg) produced broad-spectrum limbic and severe sustained seizures in all of the treated mice. The seizures were abolished when alpha-ketoglutarate (2 g/kg) or oxaloacetate (1 g/kg) was injected intraperitoneally in the animals 1 min before kainic acid administration. In addition, the administration of kainic acid caused damage to mtDNA in brain frontal and middle cortex of mice. These effects were completely abolished by the ip preinjection of alpha-ketoglutarate (2 g/kg) or oxaloacetate (1 g/kg). In vitro exposure of kainic acid (0.25, 0.5 or 1.0 mM) to brain homogenate inflicted damage to mtDNA in a concentration-dependent manner. The damage of mtDNA induced by 1.0 mM kainic acid was attenuated by the co-treatment with alpha-ketoglutarate (2.5 or 5.0 mM) or oxaloacetate (0.75 or 1.0 mM). Furthermore, in vivo and in vitro exposure of kainic acid elicited an increase in lipid peroxidation. However, the increased lipid peroxidation was completely inhibited by cotreatment of alpha-ketoglutarate or oxaloacetate. These results suggest that alpha-keto acids such as alpha-ketoglutarate and oxaloacetate play a role in the inhibition of seizures and subsequent mtDNA damage induced by the excitotoxic/neurotoxic agent, kainic acid.

A new family of satellite DNA sequences as a major component of centromeric heterochromatin in owls (Strigiformes).

PubMed

Yamada, Kazuhiko; Nishida-Umehara, Chizuko; Matsuda, Yoichi

2004-03-01

We isolated a new family of satellite DNA sequences from HaeIII- and EcoRI-digested genomic DNA of the Blakiston's fish owl ( Ketupa blakistoni). The repetitive sequences were organized in tandem arrays of the 174 bp element, and localized to the centromeric regions of all macrochromosomes, including the Z and W chromosomes, and microchromosomes. This hybridization pattern was consistent with the distribution of C-band-positive centromeric heterochromatin, and the satellite DNA sequences occupied 10% of the total genome as a major component of centromeric heterochromatin. The sequences were homogenized between macro- and microchromosomes in this species, and therefore intraspecific divergence of the nucleotide sequences was low. The 174 bp element cross-hybridized to the genomic DNA of six other Strigidae species, but not to that of the Tytonidae, suggesting that the satellite DNA sequences are conserved in the same family but fairly divergent between the different families in the Strigiformes. Secondly, the centromeric satellite DNAs were cloned from eight Strigidae species, and the nucleotide sequences of 41 monomer fragments were compared within and between species. Molecular phylogenetic relationships of the nucleotide sequences were highly correlated with both the taxonomy based on morphological traits and the phylogenetic tree constructed by DNA-DNA hybridization. These results suggest that the satellite DNA sequence has evolved by concerted evolution in the Strigidae and that it is a good taxonomic and phylogenetic marker to examine genetic diversity between Strigiformes species.

Discovery of a Regulatory Motif for Human Satellite DNA Transcription in Response to BATF2 Overexpression.

PubMed

Bai, Xuejia; Huang, Wenqiu; Zhang, Chenguang; Niu, Jing; Ding, Wei

2016-03-01

One of the basic leucine zipper transcription factors, BATF2, has been found to suppress cancer growth and migration. However, little is known about the genes downstream of BATF2. HeLa cells were stably transfected with BATF2, then chromatin immunoprecipitation-sequencing was employed to identify the DNA motifs responsive to BATF2. Comprehensive bioinformatics analyses indicated that the most significant motif discovered as TTCCATT[CT]GATTCCATTC[AG]AT was primarily distributed among the chromosome centromere regions and mostly within human type II satellite DNA. Such motifs were able to prime the transcription of type II satellite DNA in a directional and asymmetrical manner. Consistently, satellite II transcription was up-regulated in BATF2-overexpressing cells. The present study provides insight into understanding the role of BATF2 in tumours and the importance of satellite DNA in the maintenance of genomic stability. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Satellite DNA and Transposable Elements in Seabuckthorn (Hippophae rhamnoides), a Dioecious Plant with Small Y and Large X Chromosomes.

PubMed

Puterova, Janka; Razumova, Olga; Martinek, Tomas; Alexandrov, Oleg; Divashuk, Mikhail; Kubat, Zdenek; Hobza, Roman; Karlov, Gennady; Kejnovsky, Eduard

2017-01-01

Seabuckthorn (Hippophae rhamnoides) is a dioecious shrub commonly used in the pharmaceutical, cosmetic, and environmental industry as a source of oil, minerals and vitamins. In this study, we analyzed the transposable elements and satellites in its genome. We carried out Illumina DNA sequencing and reconstructed the main repetitive DNA sequences. For data analysis, we developed a new bioinformatics approach for advanced satellite DNA analysis and showed that about 25% of the genome consists of satellite DNA and about 24% is formed of transposable elements, dominated by Ty3/Gypsy and Ty1/Copia LTR retrotransposons. FISH mapping revealed X chromosome-accumulated, Y chromosome-specific or both sex chromosomes-accumulated satellites but most satellites were found on autosomes. Transposable elements were located mostly in the subtelomeres of all chromosomes. The 5S rDNA and 45S rDNA were localized on one autosomal locus each. Although we demonstrated the small size of the Y chromosome of the seabuckthorn and accumulated satellite DNA there, we were unable to estimate the age and extent of the Y chromosome degeneration. Analysis of dioecious relatives such as Shepherdia would shed more light on the evolution of these sex chromosomes. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Karyopherin alpha 1 regulates satellite cell proliferation and survival by modulating nuclear import

PubMed Central

Choo, Hyo-Jung; Cutler, Alicia; Rother, Franziska; Bader, Michael; Pavlath, Grace K.

2016-01-01

Satellite cells are stem cells with an essential role in skeletal muscle repair. Precise regulation of gene expression is critical for proper satellite cell quiescence, proliferation, differentiation and self -renewal. Nuclear proteins required for gene expression are dependent on the nucleocytoplasmic transport machinery to access to nucleus, however little is known about regulation of nuclear transport in satellite cells. The best characterized nuclear import pathway is classical nuclear import which depends on a classical nuclear localization signal (cNLS) in a cargo protein and the heterodimeric import receptors, karyopherin alpha (KPNA) and beta (KPNB). Multiple KPNA1 paralogs exist and can differ in importing specific cNLS proteins required for cell differentiation and function. We show that transcripts for six Kpna paralogs underwent distinct changes in mouse satellite cells during muscle regeneration accompanied by changes in cNLS proteins in nuclei. Depletion of KPNA1, the most dramatically altered KPNA, caused satellite cells in uninjured muscle to prematurely activate, proliferate and undergo apoptosis leading to satellite cell exhaustion with age. Increased proliferation of satellite cells led to enhanced muscle regeneration at early stages of regeneration. In addition, we observed impaired nuclear localization of two key KPNA1 cargo proteins: p27, a cyclin-dependent kinase inhibitor associated with cell cycle control and lymphoid enhancer factor 1, a critical co-transcription factor for β-catenin. These results indicate that regulated nuclear import of proteins by KPNA1 is critical for satellite cell proliferation and survival and establish classical nuclear import as a novel regulatory mechanism for controlling satellite cell fate. PMID:27434733

Distribution and sequence homogeneity of an abundant satellite DNA in the beetle, Tenebrio molitor.

PubMed Central

Davis, C A; Wyatt, G R

1989-01-01

The mealworm beetle, Tenebrio molitor, contains an unusually abundant and homogeneous satellite DNA which constitutes up to 60% of its genome. The satellite DNA is shown to be present in all of the chromosomes by in situ hybridization. 18 dimers of the repeat unit were cloned and sequenced. The consensus sequence is 142 nt long and lacks any internal repeat structure. Monomers of the sequence are very similar, showing on average a 2% divergence from the calculated consensus. Variant nucleotides are scattered randomly throughout the sequence although some variants are more common than others. Neighboring repeat units are no more alike than randomly chosen ones. The results suggest that some mechanism, perhaps gene conversion, is acting to maintain the homogeneity of the satellite DNA despite its abundance and distribution on all of the chromosomes. Images PMID:2762148

Effect of 2',3'-dideoxythymidine-5'-triphosphate on HeLa cell in vitro DNA synthesis: evidence that DNA polymerase alpha is the only polymerase required for cellular DNA replication.

PubMed Central

Waqar, M A; Evans, M J; Huberman, J A

1978-01-01

We have studied the effects of the nucleotide analogue, 2',3'-dideoxythymidine-5'-triphosphate (ddTTP) on replicative DNA synthesis in HeLa cell lysates. As previously demonstrated (1), such lysates carry out extensive DNA synthesis in vitro, at rates and in a fashion similar to in vivo DNA replication. We report here that all aspects of DNA synthesis in such lysates (total dNTP incorporation, elongation of continuous nascent strands, and the initiation, elongation, and joining of Okazaki pieces) are only slightly inhibited by concentrations of ddTTP as high as 100-500 micrometer when the dTTP concentration is maintained at 10 micrometer. This finding is consistent with the report by Edenberg, Anderson, and DePamphilis (2) that all aspects of replicative in vitro simian virus 40 DNA synthesis are also resistant to ddTTP. We also find, in agreement with Edenberg, Anderson, and DePamphilis (2), that DNA synthesis catalyzed by DNA polymerases beta or gamma is easily inhibited by ddTTP, while synthesis catalyzed by DNA polymerase alpha is very resistant. These observations suggest that DNA polymerase alpha may be the only DNA polymerase required for all aspects of cellular DNA synthesis. PMID:673840

Improved Silica-Guanidiniumthiocyanate DNA Isolation Procedure Based on Selective Binding of Bovine Alpha-Casein to Silica Particles

PubMed Central

Boom, René; Sol, Cees; Beld, Marcel; Weel, Jan; Goudsmit, Jaap; Wertheim-van Dillen, Pauline

1999-01-01

DNA purified from clinical cerebrospinal fluid and urine specimens by a silica-guanidiniumthiocyanate procedure frequently contained an inhibitor(s) of DNA-processing enzymes which may have been introduced by the purification procedure itself. Inhibition could be relieved by the use of a novel lysis buffer containing alpha-casein. When the novel lysis buffer was used, alpha-casein was bound by the silica particles in the first step of the procedure and eluted together with DNA in the last step, after which it exerted its beneficial effects for DNA-processing enzymes. In the present study we have compared the novel lysis buffer with the previously described lysis buffer with respect to double-stranded DNA yield (which was nearly 100%) and the performance of DNA-processing enzymes. PMID:9986822

Humidity influenced capacitance and resistance of an Al/DNA/Al Schottky diode irradiated by alpha particles

PubMed Central

Al-Ta’ii, Hassan Maktuff Jaber; Amin, Yusoff Mohd; Periasamy, Vengadesh

2016-01-01

Deoxyribonucleic acid or DNA based sensors, especially as humidity and alpha particle sensors have become quite popular in recent times due to flexible and highly optimizable nature of this fundamental biomaterial. Application of DNA electronics allow for more sensitive, accurate and effective sensors to be developed and fabricated. In this work, we examined the effect of different humidity conditions on the capacitive and resistive response of Aluminum (Al)/DNA/Al Schottky barrier structure when bombarded by time-dependent dosages of alpha particles. Based on current-voltage profiles, which demonstrated rectifying behaviours, Schottky diode parameters such as ideality factor, barrier height and series resistance was calculated. Results observed generally pointed towards a decrease in the resistance value from the pristine to the radiated structures. It was also demonstrated that under the effect of humidity, the capacitance of the DNA thin film increased from 0.05894 to 92.736 nF, with rising relative humidity level. We also observed the occurrence of the hypersensitivity phenomena after alpha irradiation between 2 to 4 min by observing a drop in the series resistance, crucial in the study of DNA damage and repair mechanisms. These observations may also suggest the exciting possibility of utilizing Al/DNA/Al Schottky diodes as potentially sensitive humidity sensors. PMID:27160654

cDNA cloning and characterization of Type I procollagen alpha1 chain in the skate Raja kenojei.

PubMed

Hwang, Jae-Ho; Yokoyama, Yoshihiro; Mizuta, Shoshi; Yoshinaka, Reiji

2006-05-01

A full-length cDNA of the Type I procollagen alpha1 [pro-alpha1(I)] chain (4388 bp), coding for 1463 amino acid residues in the total length, was determined by RACE PCR using a cDNA library constructed from 4-week embryo of the skate Raja kenojei. The helical region of the skate pro-alpha1(I) chain consisted of 1014 amino acid residues - the same as other fibrillar collagen alpha chains from higher vertebrates. Comparison on denaturation temperatures of Type I collagens from the skate, rainbow trout (Oncorhynchus mykiss) and rat (Rattus norvegicus) revealed that the number of Gly-Pro-Pro and Gly-Gly in the alpha1(I) chains could be directly related to the thermal stability of the helix. The expression property of the skate pro-alpha1(I) chain mRNA and phylogenetic analysis with other vertebrate pro-alpha1(I) chains suggested that skate pro-alpha1(I) chain could be a precursor form of the skate Type I collagen alpha1 chain. The present study is the first evidence for the primary structure of full-length pro-alpha1(I) chain in an elasmobranch.

Induction of single- and double-strand breaks in plasmid DNA by monoenergetic alpha-particles with energies below the Bragg-maximum.

PubMed

Scholz, V; Weidner, J; Köhnlein, W; Frekers, D; Wörtche, H J

1997-01-01

The yield of single-strand breaks (ssb) and double-strand breaks (dsb) produced by alpha-particles at the end of their track in DNA-films was determined experimentally. Helium nuclei were accelerated to 600 keV in the 400 kV ion accelerator and scattered at a carbon target. The elastically scattered alpha-particles with energies of 344 keV and 485 keV were used to irradiate supercircular plasmid DNA in vacuo. For the dosimetry of the alpha-particles a surface barrier detector was used and the energy distribution of the alpha-particles determined. The energy loss of the particles in the DNA-layer was calculated. DNA samples were separated into the three conformational isomers using agarose gel electrophoresis. After fluorochromation the number of ssb and dsb per plasmid DNA molecule was established from the band intensities assuming the validity of Poisson statistics. Linear dose effect correlations were found for ssb and dsb per plasmid molecule. In the case of 344 keV-alpha-particles the yield of dsb was (8.6 +/- 0.9) x 10(-11) breaks/Gy x dalton. The ratio of ssb/dsb was 0.5 +/- 0.2. This is at least a factor of six larger than the ratio found in experiments with higher energy alpha-particles and from model calculations. Similar experiments with protons yielded a relative biological effectiveness (rbe) value of 2.8 for the induction of double-strand breaks by track end alpha-particles.

Electronic Properties of DNA-Based Schottky Barrier Diodes in Response to Alpha Particles.

PubMed

Al-Ta'ii, Hassan Maktuff Jaber; Periasamy, Vengadesh; Amin, Yusoff Mohd

2015-05-21

Detection of nuclear radiation such as alpha particles has become an important field of research in recent history due to nuclear threats and accidents. In this context; deoxyribonucleic acid (DNA) acting as an organic semiconducting material could be utilized in a metal/semiconductor Schottky junction for detecting alpha particles. In this work we demonstrate for the first time the effect of alpha irradiation on an Al/DNA/p-Si/Al Schottky diode by investigating its current-voltage characteristics. The diodes were exposed for different periods (0-20 min) of irradiation. Various diode parameters such as ideality factor, barrier height, series resistance, Richardson constant and saturation current were then determined using conventional, Cheung and Cheung's and Norde methods. Generally, ideality factor or n values were observed to be greater than unity, which indicates the influence of some other current transport mechanism besides thermionic processes. Results indicated ideality factor variation between 9.97 and 9.57 for irradiation times between the ranges 0 to 20 min. Increase in the series resistance with increase in irradiation time was also observed when calculated using conventional and Cheung and Cheung's methods. These responses demonstrate that changes in the electrical characteristics of the metal-semiconductor-metal diode could be further utilized as sensing elements to detect alpha particles.

Amplification of the major satellite DNA family (FA-SAT) in a cat fibrosarcoma might be related to chromosomal instability.

PubMed

Santos, Sara; Chaves, Raquel; Adega, Filomena; Bastos, Estela; Guedes-Pinto, Henrique

2006-01-01

Most mammalian chromosomes have satellite DNA sequences located at or near the centromeres, organized in arrays of variable size and higher order structure. The implications of these specific repetitive DNA sequences and their organization for centromere function are still quite cloudy. In contrast to most mammalian species, the domestic cat seems to have the major satellite DNA family (FA-SAT) localized primarily at the telomeres and secondarily at the centromeres of the chromosomes. In the present work, we analyzed chromosome preparations from a fibrosarcoma, in comparison with nontumor cells (epithelial tissue) from the same individual, by in situ hybridization of the FA-SAT cat satellite DNA family. This repetitive sequence was found to be amplified in the cat tumor chromosomes analyzed. The amplification of these satellite DNA sequences in the cat chromosomes with variable number and appearance (marker chromosomes) is discussed and might be related to mitotic instability, which could explain the exhibition of complex patterns of chromosome aberrations detected in the fibrosarcoma analyzed.

Characterization of Non-coding DNA Satellites Associated with Sweepoviruses (Genus Begomovirus, Geminiviridae) – Definition of a Distinct Class of Begomovirus-Associated Satellites

PubMed Central

Lozano, Gloria; Trenado, Helena P.; Fiallo-Olivé, Elvira; Chirinos, Dorys; Geraud-Pouey, Francis; Briddon, Rob W.; Navas-Castillo, Jesús

2016-01-01

Begomoviruses (family Geminiviridae) are whitefly-transmitted, plant-infecting single-stranded DNA viruses that cause crop losses throughout the warmer parts of the World. Sweepoviruses are a phylogenetically distinct group of begomoviruses that infect plants of the family Convolvulaceae, including sweet potato (Ipomoea batatas). Two classes of subviral molecules are often associated with begomoviruses, particularly in the Old World; the betasatellites and the alphasatellites. An analysis of sweet potato and Ipomoea indica samples from Spain and Merremia dissecta samples from Venezuela identified small non-coding subviral molecules in association with several distinct sweepoviruses. The sequences of 18 clones were obtained and found to be structurally similar to tomato leaf curl virus-satellite (ToLCV-sat, the first DNA satellite identified in association with a begomovirus), with a region with significant sequence identity to the conserved region of betasatellites, an A-rich sequence, a predicted stem–loop structure containing the nonanucleotide TAATATTAC, and a second predicted stem–loop. These sweepovirus-associated satellites join an increasing number of ToLCV-sat-like non-coding satellites identified recently. Although sharing some features with betasatellites, evidence is provided to suggest that the ToLCV-sat-like satellites are distinct from betasatellites and should be considered a separate class of satellites, for which the collective name deltasatellites is proposed. PMID:26925037

Satellite DNA-based artificial chromosomes for use in gene therapy.

PubMed

Hadlaczky, G

2001-04-01

Satellite DNA-based artificial chromosomes (SATACs) can be made by induced de novo chromosome formation in cells of different mammalian species. These artificially generated accessory chromosomes are composed of predictable DNA sequences and they contain defined genetic information. Prototype human SATACs have been successfully constructed in different cell types from 'neutral' endogenous DNA sequences from the short arm of the human chromosome 15. SATACs have already passed a number of hurdles crucial to their further development as gene therapy vectors, including: large-scale purification; transfer of purified artificial chromosomes into different cells and embryos; generation of transgenic animals and germline transmission with purified SATACs; and the tissue-specific expression of a therapeutic gene from an artificial chromosome in the milk of transgenic animals.

Centromeric DNA replication reconstitution reveals DNA loops and ATR checkpoint suppression.

PubMed

Aze, Antoine; Sannino, Vincenzo; Soffientini, Paolo; Bachi, Angela; Costanzo, Vincenzo

2016-06-01

Half of the human genome is made up of repetitive DNA. However, mechanisms underlying replication of chromosome regions containing repetitive DNA are poorly understood. We reconstituted replication of defined human chromosome segments using bacterial artificial chromosomes in Xenopus laevis egg extract. Using this approach we characterized the chromatin assembly and replication dynamics of centromeric alpha-satellite DNA. Proteomic analysis of centromeric chromatin revealed replication-dependent enrichment of a network of DNA repair factors including the MSH2-6 complex, which was required for efficient centromeric DNA replication. However, contrary to expectations, the ATR-dependent checkpoint monitoring DNA replication fork arrest could not be activated on highly repetitive DNA due to the inability of the single-stranded DNA binding protein RPA to accumulate on chromatin. Electron microscopy of centromeric DNA and supercoil mapping revealed the presence of topoisomerase I-dependent DNA loops embedded in a protein matrix enriched for SMC2-4 proteins. This arrangement suppressed ATR signalling by preventing RPA hyper-loading, facilitating replication of centromeric DNA. These findings have important implications for our understanding of repetitive DNA metabolism and centromere organization under normal and stressful conditions.

Centromeric DNA replication reconstitution reveals DNA loops and ATR checkpoint suppression

PubMed Central

Aze, Antoine; Sannino, Vincenzo; Soffientini, Paolo; Bachi, Angela; Costanzo, Vincenzo

2016-01-01

Half of human genome is made of repetitive DNA. However, mechanisms underlying replication of chromosome regions containing repetitive DNA are poorly understood. We reconstituted replication of defined human chromosome segments using Bacterial Artificial Chromosomes (BACs) in Xenopus laevis egg extract. Using this approach we characterized chromatin assembly and replication dynamics of centromeric alpha-satellite DNA. Proteomic analysis of centromeric chromatin revealed replication dependent enrichment of a network of DNA repair factors among which the MSH2-6 complex, which was required for efficient centromeric DNA replication. However, contrary to expectations, the ATR dependent checkpoint monitoring DNA replication fork arrest could not be activated on highly repetitive DNA due to inability of single stranded DNA binding protein RPA to accumulate on chromatin. Electron microscopy of centromeric DNA and supercoil mapping revealed the presence of Topoisomerase I dependent DNA loops embedded in a protein matrix enriched for SMC2-4 proteins. This arrangement suppressed ATR signalling by preventing RPA hyper-loading, facilitating replication of centromeric DNA. These findings have important implications on our understanding of repetitive DNA metabolism and centromere organization under normal and stressful conditions. PMID:27111843

FA-SAT Is an Old Satellite DNA Frozen in Several Bilateria Genomes

PubMed Central

Chaves, Raquel; Ferreira, Daniela; Mendes-da-Silva, Ana; Meles, Susana; Adega, Filomena

2017-01-01

Abstract In recent years, a growing body of evidence has recognized the tandem repeat sequences, and specifically satellite DNA, as a functional class of sequences in the genomic “dark matter.” Using an original, complementary, and thus an eclectic experimental design, we show that the cat archetypal satellite DNA sequence, FA-SAT, is “frozen” conservatively in several Bilateria genomes. We found different genomic FA-SAT architectures, and the interspersion pattern was conserved. In Carnivora genomes, the FA-SAT-related sequences are also amplified, with the predominance of a specific FA-SAT variant, at the heterochromatic regions. We inspected the cat genome project to locate FA-SAT array flanking regions and revealed an intensive intermingling with transposable elements. Our results also show that FA-SAT-related sequences are transcribed and that the most abundant FA-SAT variant is not always the most transcribed. We thus conclude that the DNA sequences of FA-SAT and their transcripts are “frozen” in these genomes. Future work is needed to disclose any putative function that these sequences may play in these genomes. PMID:29608678

Insights on genome size evolution from a miniature inverted repeat transposon driving a satellite DNA.

PubMed

Scalvenzi, Thibault; Pollet, Nicolas

2014-12-01

The genome size in eukaryotes does not correlate well with the number of genes they contain. We can observe this so-called C-value paradox in amphibian species. By analyzing an amphibian genome we asked how repetitive DNA can impact genome size and architecture. We describe here our discovery of a Tc1/mariner miniature inverted-repeat transposon family present in Xenopus frogs. These transposons named miDNA4 are unique since they contain a satellite DNA motif. We found that miDNA4 measured 331 bp, contained 25 bp long inverted terminal repeat sequences and a sequence motif of 119 bp present as a unique copy or as an array of 2-47 copies. We characterized the structure, dynamics, impact and evolution of the miDNA4 family and its satellite DNA in Xenopus frog genomes. This led us to propose a model for the evolution of these two repeated sequences and how they can synergize to increase genome size. Copyright © 2014 Elsevier Inc. All rights reserved.

RRR-alpha-tocopheryl succinate inhibits EL4 thymic lymphoma cell growth by inducing apoptosis and DNA synthesis arrest.

PubMed

Yu, W; Sanders, B G; Kline, K

1997-01-01

RRR-alpha-tocopheryl succinate (vitamin E succinate, VES) treatment of murine EL4 T lymphoma cells induced the cells to undergo apoptosis. After 48 hours of VES treatment at 20 micrograms/ml, 95% of cells were apoptotic. Evidence for the induction of apoptosis by VES treatments is based on staining of DNA for detection of chromatin condensation/fragmentation, two-color flow-cytometric analyses of DNA content, and end-labeled DNA and electrophoretic analyses for detection of DNA ladder formation. VES-treated EL4 cells were blocked in the G1 cell cycle phase; however, apoptotic cells came from all cell cycle phases. Analyses of mRNA expression of genes involved in apoptosis revealed decreased c-myc and increased bcl-2, c-fos, and c-jun mRNAs within three to six hours after treatment. Western analyses showed increased c-Jun, c-Fos, and Bcl-2 protein levels. Electrophoretic mobility shift assays showed increased AP-1 binding at 6, 12, and 24 hours after treatment and decreased c-Myc binding after 12 and 24 hours of VES treatment. Treatments of EL4 cells with VES+RRR-alpha-to-copherol reduced apoptosis without effecting DNA synthesis arrest. Treatments of EL4 cells with VES+rac-6-hydroxyl-2, 5,7,8-tetramethyl-chroman-2-carboxylic acid, butylated hydroxytoluene, or butylated hydroxyanisole had no effect on apoptosis or DNA synthesis arrest caused by VES treatments. Analyses of bcl-2, c-myc, c-jun, and c-fos mRNA levels in cells receiving VES + RRR-alpha-tocopherol treatments showed no change from cells receiving VES treatments alone, implying that these changes are correlated with VES treatments but are not causal for apoptosis. However, treatments with VES + RRR-alpha-tocopherol decreased AP-1 binding to consensus DNA oligomer, suggesting AP-1 involvement in apoptosis induced by VES treatments.

Concerted evolution at the population level: pupfish HindIII satellite DNA sequences.

PubMed Central

Elder, J F; Turner, B J

1994-01-01

The canonical monomers (approximately 170 bp) of an abundant (1.9 x 10(6) copies per diploid genome) satellite DNA sequence family in the genome of Cyprinodon variegatus, a "pupfish" that ranges along the Atlantic coast from Cape Cod to central Mexico, are divergent in base sequence in 10 of 12 samples collected from natural populations. The divergence involves substitutions, deletions, and insertions, is marked in scope (mean pairwise sequence similarity = 61.6%; range = 35-95.9%), is largely confined to the 3' half of the monomer, and is not correlated with the distance among collecting sites. Repetitive cloning and direct genomic sequencing experiments failed to detect intrapopulation and intraindividual variation, suggesting high levels of sequence homogeneity within populations. The satellite sequence has therefore undergone "concerted evolution," at the level of the local population. Concerted evolution has previously almost always been discussed in terms of the divergence of species or higher taxa; its intraspecific occurrence apparently has not been reported previously. The generality of the observation is difficult to evaluate, for although satellite DNAs from a large number of organisms have been studied in detail, there appear to be little or no other data on their sequence variation in natural populations. The relationship (if any) between concerted, population level, satellite DNA divergence and the extent of gene flow/genetic isolation among conspecific natural populations remains to be established. Images PMID:8302879

A new mechanism for DNA alterations induced by alpha particles such as those emitted by radon and radon progeny.

PubMed Central

Lehnert, B E; Goodwin, E H

1997-01-01

The mechanism(s) by which alpha (alpha) particles like those emitted from inhaled radon and radon progeny cause their carcinogenic effects in the lung remains unclear. Although direct nuclear traversals by alpha-particles may be involved in mediating these outcomes, increasing evidence indicates that a particles can cause alterations in DNA in the absence of direct hits to cell nuclei. Using the occurrence of excessive sister chromatid exchanges (SCE) as an index of DNA damage in human lung fibroblasts, we investigated the hypothesis that alpha-particles may induce DNA damage through the generation of extracellular factors. We have found that a relatively low dose of alpha-particles can result in the generation of extracellular factors, which, upon transfer to unexposed normal human cells, can cause excessive SCE to an extent equivalent to that observed when the cells are directly irradiated with the same irradiation dose. A short-lived, SCE-inducing factor(s) is generated in alpha-irradiated culture medium containing serum in the absence of cells. A more persistent SCE-inducing factor(s), which can survive freeze-thaw and is heat labile is produced by fibroblasts after exposure to the alpha-particles. These results indicate that the initiating target for alpha-particle-induced genetic changes can be larger than a cell's nucleus or even a whole cell. How transmissible factors like those observed here in vitro may extend to the in vivo condition in the context of a-particle-induced carcinogenesis in the respiratory tract remains to be determined. PMID:9400706

Satellite RNA Increases DNA Damage and Accelerates Tumor Formation in Mouse Models of Pancreatic Cancer.

PubMed

Kishikawa, Takahiro; Otsuka, Motoyuki; Suzuki, Tatsunori; Seimiya, Takahiro; Sekiba, Kazuma; Ishibashi, Rei; Tanaka, Eri; Ohno, Motoko; Yamagami, Mari; Koike, Kazuhiko

2018-05-10

Highly repetitive tandem arrays such as satellite sequences in the centromeric and pericentromeric regions of chromosomes, which were previously considered to be silent, are actively transcribed in various biological processes, including cancers. In the pancreas, this aberrant expression occurs even in Kras-mutated pancreatic intraepithelial neoplasia (PanIN) tissues, which are precancerous lesions. To determine the biological role of satellite RNAs in carcinogenesis in vivo , we constructed mouse major satellite (MajSAT) RNA-expressing transgenic mice. However, these transgenic mice did not show spontaneous malignant tumor formation under normal breeding. Importantly, however, DNA damage was increased in pancreatic tissues induced by caerulein treatment or high-fat diet, which may be due to impaired nuclear localization of Y-Box Binding Protein 1 (YBX1), a component of the DNA damage repair machinery. In addition, when crossed with pancreas-specific Kras-mutant mice, MajSAT RNA expression resulted in an earlier increase in PanIN formation. These results suggest that aberrant MajSAT RNA expression accelerates oncogenesis by increasing the probability of a second driver mutation, thus accelerating cells to exit from the breakthrough phase to the expansion phase. Implications: Aberrant expression of satellite RNAs accelerates oncogenesis through a mechanism involving increased DNA damage. Mol Cancer Res; 1-8. ©2018 AACR. ©2018 American Association for Cancer Research.

Immunohistochemical study of DNA topoisomerase I, DNA topoisomerase II alpha, p53, and Ki-67 in oral preneoplastic lesions and oral squamous cell carcinomas.

PubMed

Hafian, Hilal; Venteo, Lydie; Sukhanova, Alyona; Nabiev, Igor; Lefevre, Benoît; Pluot, Michel

2004-06-01

Human DNA topoisomerase I (topo I) is the molecular target of the camptothecin group of anticancer drugs. Laboratory studies have shown that the cellular response to topo I-targeted drugs depends on the topo I expression and DNA replication rate and the apoptotic pathway activity. In this study, we tested potential indicators of the sensitivity of topo I-targeted drugs in 36 cases of oral squamous cell carcinoma (OSCC). Formalin-fixed, paraffin-embedded tissue sections were immunostained with monoclonal antibodies against Ki-67, p53, and topo I, and with polyclonal antibodies against DNA topoisomerase II-alpha (topo II-alpha). These markers were also tested in 18 epithelial hyperplastic lesions and 18 mild dysplasias. Immunostaining was quantified by the percentage of stained nuclei in each sample (the labeling index); 200 immunoreactive epithelial nuclei were counted per case for each antibody. The results support the possibility of using topo II-alpha staining for assessing the proliferative activity. High expression of topo II-alpha and topo I in OSCCs suggests that they may serve as potential indicators of sensitivity to topo I inhibitors. However, the apoptotic pathway assessed by p53 immunostaining was found to be uninformative. Analysis of the relationship between immunohistochemical results and clinical and pathologic parameters (the T and N stages and differentiation) showed that only the differentiation parameter correlated with the topo I expression rate. Thus, significant increase in the topo I expression in the poorly differentiated OSCCs suggests their higher sensitivity to drug treatment.

Individual specific DNA fingerprints from a hypervariable region probe: alpha-globin 3'HVR.

PubMed

Fowler, S J; Gill, P; Werrett, D J; Higgs, D R

1988-06-01

A probe detecting a hypervariable region (HVR) 3' to the alpha globin locus on chromosome 16 has been used to produce DNA fingerprints. Segregation analysis has revealed multiple, randomly dispersed DNA fragments inherited in a Mendelian fashion with minimal allelism and linkage. The fingerprints are highly polymorphic (probability of chance association between random individuals much less than 10(-14]. The probe is, therefore, a powerful discriminating tool: it is envisaged that this probe will have forensic applications, including paternity cases, and will be informative in linkage analysis.

Evolutionary Story of a Satellite DNA from Phodopus sungorus (Rodentia, Cricetidae)

PubMed Central

Paço, Ana; Adega, Filomena; Meštrović, Nevenka; Plohl, Miroslav; Chaves, Raquel

2014-01-01

With the goal to contribute for the understanding of satellite DNA evolution and its genomic involvement, in this work it was isolated and characterized the first satellite DNA (PSUcentSat) from Phodopus sungorus (Cricetidae). Physical mapping of this sequence in P. sungorus showed large PSUcentSat arrays located at the heterochromatic (peri)centromeric region of five autosomal pairs and Y-chromosome. The presence of orthologous PSUcentSat sequences in the genomes of other Cricetidae and Muridae rodents was also verified, presenting however, an interspersed chromosomal distribution. This distribution pattern suggests a PSUcentSat-scattered location in an ancestor of Muridae/Cricetidae families, that assumed afterwards, in the descendant genome of P. sungorus a restricted localization to few chromosomes in the (peri)centromeric region. We believe that after the divergence of the studied species, PSUcentSat was most probably highly amplified in the (peri)centromeric region of some chromosome pairs of this hamster by recombinational mechanisms. The bouquet chromosome configuration (prophase I) possibly displays an important role in this selective amplification, providing physical proximity of centromeric regions between chromosomes with similar size and/or morphology. This seems particularly evident for the acrocentric chromosomes of P. sungorus (including the Y-chromosome), all presenting large PSUcentSat arrays at the (peri)centromeric region. The conservation of this sequence in the studied genomes and its (peri)centromeric amplification in P. sungorus strongly suggests functional significance, possibly displaying this satellite family different functions in the different genomes. The verification of PSUcentSat transcriptional activity in normal proliferative cells suggests that its transcription is not stage-limited, as described for some other satellites. PMID:25336681

Is radon emission in caves causing deletions in satellite DNA sequences of cave-dwelling crickets?

PubMed

Allegrucci, Giuliana; Sbordoni, Valerio; Cesaroni, Donatella

2015-01-01

The most stable isotope of radon, 222Rn, represents the major source of natural radioactivity in confined environments such as mines, caves and houses. In this study, we explored the possible radon-related effects on the genome of Dolichopoda cave crickets (Orthoptera, Rhaphidophoridae) sampled in caves with different concentrations of radon. We analyzed specimens from ten populations belonging to two genetically closely related species, D. geniculata and D. laetitiae, and explored the possible association between the radioactivity dose and the level of genetic polymorphism in a specific family of satellite DNA (pDo500 satDNA). Radon concentration in the analyzed caves ranged from 221 to 26,000 Bq/m3. Specimens coming from caves with the highest radon concentration showed also the highest variability estimates in both species, and the increased sequence heterogeneity at pDo500 satDNA level can be explained as an effect of the mutation pressure induced by radon in cave. We discovered a specific category of nuclear DNA, the highly repetitive satellite DNA, where the effects of the exposure at high levels of radon-related ionizing radiation are detectable, suggesting that the satDNA sequences might be a valuable tool to disclose harmful effects also in other organisms exposed to high levels of radon concentration.

alpha-Putrescinylthymine and the sensitivity of bacteriophage phi W-14 DNA to restriction endonucleases.

PubMed Central

Miller, P B; Wakarchuk, W W; Warren, R A

1985-01-01

The modified base alpha-putrescinylthymine (putT) in phi W-14 DNA blocks cleavage of the DNA by 17 of 32 Type II restriction endonucleases. The enzymes cleaving the DNA do so to widely varying extents. The frequencies of cleavage of three altered forms of the DNA show that putT blocks recognition sites either when it occurs within the site or when it is in a sequence flanking the site. The blocking is dependent on both charge and steric factors. The charge effects can be greater than the steric effects for some of the enzymes tested. All the enzymes cleaving phi W-14 DNA release discrete fragments, showing that the distribution of putT is ordered. The cleavage frequencies for different enzymes suggest that the sequence CAputTG occurs frequently in the DNA. Only TaqI of the enzymes tested appeared not to be blocked by putT, but it was slowed down. TaqI generated fragments are joinable by T4 DNA ligase. PMID:2987859

Ordered mapping of 3 alphoid DNA subsets on human chromosome 22

DOE Office of Scientific and Technical Information (OSTI.GOV)

Antonacci, R.; Baldini, A.; Archidiacono, N.

1994-09-01

Alpha satellite DNA consists of tandemly repeated monomers of 171 bp clustered in the centromeric region of primate chromosomes. Sequence divergence between subsets located in different human chromosomes is usually high enough to ensure chromosome-specific hybridization. Alphoid probes specific for almost every human chromosome have been reported. A single chromosome can carry different subsets of alphoid DNA and some alphoid subsets can be shared by different chromosomes. We report the physical order of three alphoid DNA subsets on human chromosome 22 determined by a combination of low and high resolution cytological mapping methods. Results visually demonstrate the presence of threemore » distinct alphoid DNA domains at the centromeric region of chromosome 22. We have measured the interphase distances between the three probes in three-color FISH experiments. Statistical analysis of the results indicated the order of the subsets. Two color experiments on prometaphase chromosomes established the order of the three domains relative to the arms of chromosome 22 and confirmed the results obtained using interphase mapping. This demonstrates the applicability of interphase mapping for alpha satellite DNA orderering. However, in our experiments, interphase mapping did not provide any information about the relationship between extremities of the repeat arrays. This information was gained from extended chromatin hybridization. The extremities of two of the repeat arrays were seen to be almost overlapping whereas the third repeat array was clearly separated from the other two. Our data show the value of extended chromatin hybridization as a complement of other cytological techniques for high resolution mapping of repetitive DNA sequences.« less

Thermodynamic characterization of binding Oxytricha nova single strand telomere DNA with the alpha protein N-terminal domain.

PubMed

Buczek, Pawel; Horvath, Martin P

2006-06-23

The Oxytricha nova telemere binding protein alpha subunit binds single strand DNA and participates in a nucleoprotein complex that protects the very ends of chromosomes. To understand how the N-terminal, DNA binding domain of alpha interacts with DNA we measured the stoichiometry, enthalpy (DeltaH), entropy (DeltaS), and dissociation constant (K(D-DNA)) for binding telomere DNA fragments at different temperatures and salt concentrations using native gel electrophoresis and isothermal titration calorimetry (ITC). About 85% of the total free energy of binding corresponded with non-electrostatic interactions for all DNAs. Telomere DNA fragments d(T(2)G(4)), d(T(4)G(4)), d(G(3)T(4)G(4)), and d(G(4)T(4)G(4)) each formed monovalent protein complexes. In the case of d(T(4)G(4)T(4)G(4)), which has two tandemly repeated d(TTTTTGGGG) telomere motifs, two binding sites were observed. The high-affinity "A site" has a dissociation constant, K(D-DNA(A)) = 13(+/-4) nM, while the low-affinity "B site" is characterized by K(D-DNA(B)) = 5600(+/-600) nM at 25 degrees C. Nucleotide substitution variants verified that the A site corresponds principally with the 3'-terminal portion of d(T(4)G(4)T(4)G(4)). The relative contributions of entropy (DeltaS) and enthalpy (DeltaH) for binding reactions were DNA length-dependent as was heat capacity (DeltaCp). These trends with respect to DNA length likely reflect structural transitions in the DNA molecule that are coupled with DNA-protein association. Results presented here are important for understanding early intermediates and subsequent stages in the assembly of the full telomere nucleoprotein complex and how binding events can prepare the telomere DNA for extension by telomerase, a critical event in telomere biology.

Thermodynamic Characterization of Binding Oxytricha nova Single Strand Telomere DNA with the Alpha Protein N-terminal Domain

PubMed Central

Buczek, Pawel; Horvath, Martin P.

2010-01-01

The Oxytricha nova telomere binding protein alpha subunit binds single strand DNA and participates in a nucleoprotein complex that protects the very ends of chromosomes. To understand how the N-terminal, DNA binding domain of alpha interacts with DNA we measured the stoichiometry, enthalpy (ΔH), entropy (ΔS), and dissociation constant (KD-DNA) for binding telomere DNA fragments at different temperatures and salt concentrations using native gel electrophoresis and isothermal titration calorimetry (ITC). About 85% of the total free energy of binding corresponded with non-electrostatic interactions for all DNAs. Telomere DNA fragments d(T2G4), d(T4G4), d(G3T4G4), and d(G4T4G4) each formed monovalent protein complexes. In the case of d(T4G4T4G4), which has two tandemly repeated d(TTTTTGGGG) telomere motifs, two binding sites were observed. The high-affinity “A site” has a dissociation constant, KD-DNA(A)=13(±4) nM, while the low-affinity “B site” is characterized by KD-DNA(B)=5600(±600) nM at 25 °C. Nucleotide substitution variants verified that the A site corresponds principally with the 3′-terminal portion of d(T4G4T4G4). The relative contributions of entropy (ΔS) and enthalpy (ΔH) for binding reactions were DNA length-dependent as was heat capacity (ΔCp). These trends with respect to DNA length likely reflect structural transitions in the DNA molecule that are coupled with DNA–protein association. Results presented here are important for understanding early intermediates and subsequent stages in the assembly of the full telomere nucleoprotein complex and how binding events can prepare the telomere DNA for extension by telomerase, a critical event in telomere biology. PMID:16678852

A Glimpse into the Satellite DNA Library in Characidae Fish (Teleostei, Characiformes)

PubMed Central

Utsunomia, Ricardo; Ruiz-Ruano, Francisco J.; Silva, Duílio M. Z. A.; Serrano, Érica A.; Rosa, Ivana F.; Scudeler, Patrícia E. S.; Hashimoto, Diogo T.; Oliveira, Claudio; Camacho, Juan Pedro M.; Foresti, Fausto

2017-01-01

Satellite DNA (satDNA) is an abundant fraction of repetitive DNA in eukaryotic genomes and plays an important role in genome organization and evolution. In general, satDNA sequences follow a concerted evolutionary pattern through the intragenomic homogenization of different repeat units. In addition, the satDNA library hypothesis predicts that related species share a series of satDNA variants descended from a common ancestor species, with differential amplification of different satDNA variants. The finding of a same satDNA family in species belonging to different genera within Characidae fish provided the opportunity to test both concerted evolution and library hypotheses. For this purpose, we analyzed here sequence variation and abundance of this satDNA family in ten species, by a combination of next generation sequencing (NGS), PCR and Sanger sequencing, and fluorescence in situ hybridization (FISH). We found extensive between-species variation for the number and size of pericentromeric FISH signals. At genomic level, the analysis of 1000s of DNA sequences obtained by Illumina sequencing and PCR amplification allowed defining 150 haplotypes which were linked in a common minimum spanning tree, where different patterns of concerted evolution were apparent. This also provided a glimpse into the satDNA library of this group of species. In consistency with the library hypothesis, different variants for this satDNA showed high differences in abundance between species, from highly abundant to simply relictual variants. PMID:28855916

High dosage of ascorbic acid and alpha-tocopherol is not useful for diminishing oxidative stress and DNA damage in healthy elderly adults.

PubMed

Retana-Ugalde, Raquel; Casanueva, Esther; Altamirano-Lozano, Mario; González-Torres, Cristina; Mendoza-Núñez, Víctor Manuel

2008-01-01

To determine the useful dosage of ascorbic acid and alpha-tocopherol against oxidative stress and DNA damage in the elderly. A double-blind controlled clinical assay carried out in a sample of 66 healthy subjects divided into three age-paired random groups with 22 subjects in each group. Group A received placebo and group B was administered 500 mg of ascorbic acid and 400 IU of alpha-tocopherol, whereas group C received 1,000 mg of ascorbic acid and 400 IU of alpha-tocopherol for a 6-month period. The following measurements were performed before and after the 6-month treatment period: thiobarbituric acid reactive substances (TBARS); total antioxidant status (TAS); superoxide dismutase (SOD), and glutation peroxidase (GPx) and DNA damage by comet assay. After 6 months, group B subjects exhibited an increase in SOD and GPx enzyme levels; however, this was not statistically significant (p > 0.05). Likewise, TBARS and TAS concentrations remained unchanged (p > 0.05). In addition, in group C the decrease in TBARS and increase in SOD, GPx, and TAS were not statistically significant (p > 0.05). Similarly, average DNA migration showed no significant differences with high-dosage ascorbic acid and alpha-tocopherol. These findings suggest that administration of 1,000 mg of ascorbic acid plus 400 IU of alpha-tocopherol for 6 months is not useful for diminishing oxidative stress and DNA damage in healthy elderly adults. 2008 S. Karger AG, Basel.

The octamer-binding proteins form multi-protein--DNA complexes with the HSV alpha TIF regulatory protein.

PubMed Central

Kristie, T M; LeBowitz, J H; Sharp, P A

1989-01-01

The herpes simplex virus transactivator, alpha TIF, stimulates transcription of the alpha/immediate early genes via a cis-acting site containing an octamer element and a conserved flanking sequence. The alpha TIF protein, produced in a baculovirus expression system, nucleates the formation of at least two DNA--protein complexes on this regulatory element. Both of these complexes contain the ubiquitous Oct-1 protein, whose POU domain alone is sufficient to allow assembly of the alpha TIF-dependent complexes. A second member of the POU domain family, the lymphoid specific Oct-2 protein, can also be assembled into similar complexes at high concentrations of alpha TIF protein. These complexes contain at least two cellular proteins in addition to Oct-1. One of these proteins is present in both insect and HeLa cells and probably recognizes sequences in the cis element. The second cellular protein, only present in HeLa cells, probably binds by protein-protein interactions. Images PMID:2556266

The octamer-binding proteins form multi-protein--DNA complexes with the HSV alpha TIF regulatory protein.

PubMed

Kristie, T M; LeBowitz, J H; Sharp, P A

1989-12-20

The herpes simplex virus transactivator, alpha TIF, stimulates transcription of the alpha/immediate early genes via a cis-acting site containing an octamer element and a conserved flanking sequence. The alpha TIF protein, produced in a baculovirus expression system, nucleates the formation of at least two DNA--protein complexes on this regulatory element. Both of these complexes contain the ubiquitous Oct-1 protein, whose POU domain alone is sufficient to allow assembly of the alpha TIF-dependent complexes. A second member of the POU domain family, the lymphoid specific Oct-2 protein, can also be assembled into similar complexes at high concentrations of alpha TIF protein. These complexes contain at least two cellular proteins in addition to Oct-1. One of these proteins is present in both insect and HeLa cells and probably recognizes sequences in the cis element. The second cellular protein, only present in HeLa cells, probably binds by protein-protein interactions.

Calculation of the Electronic Parameters of an Al/DNA/p-Si Schottky Barrier Diode Influenced by Alpha Radiation

PubMed Central

Al-Ta’ii, Hassan Maktuff Jaber; Amin, Yusoff Mohd; Periasamy, Vengadesh

2015-01-01

Many types of materials such as inorganic semiconductors have been employed as detectors for nuclear radiation, the importance of which has increased significantly due to recent nuclear catastrophes. Despite the many advantages of this type of materials, the ability to measure direct cellular or biological responses to radiation might improve detector sensitivity. In this context, semiconducting organic materials such as deoxyribonucleic acid or DNA have been studied in recent years. This was established by studying the varying electronic properties of DNA-metal or semiconductor junctions when exposed to radiation. In this work, we investigated the electronics of aluminium (Al)/DNA/silicon (Si) rectifying junctions using their current-voltage (I-V) characteristics when exposed to alpha radiation. Diode parameters such as ideality factor, barrier height and series resistance were determined for different irradiation times. The observed results show significant changes with exposure time or total dosage received. An increased deviation from ideal diode conditions (7.2 to 18.0) was observed when they were bombarded with alpha particles for up to 40 min. Using the conventional technique, barrier height values were observed to generally increase after 2, 6, 10, 20 and 30 min of radiation. The same trend was seen in the values of the series resistance (0.5889–1.423 Ω for 2–8 min). These changes in the electronic properties of the DNA/Si junctions could therefore be utilized in the construction of sensitive alpha particle detectors. PMID:25730484

Acoustic satellite behaviour in the Australian bushcricket Elephantodeta nobilis (Phaneropterinae, Tettigoniidae, Orthoptera).

PubMed

Bailey; Field

2000-02-01

Male and female Elephantodeta nobilis duet with the female responding to the male's long and complex call. The duetting male's call consisted of four parts, described here as parts A, B, C and D. We found that the female replied 570 ms after the male's D pulse, which followed the extended part B and short click of part C. Noncalling males were attracted to the duet and often used satellite tactics by inserting a volley of clicks 200 ms before the alpha male's D pulse. Satellite males used part C of the alpha male song to cue their own call and this inserted call induced females to reply earlier compared with the alpha male call alone. Alpha males often extended their calls with additional D-type calls and so we examined the effectiveness of these calls as countermeasures to satellite calling. There was no influence of this alpha strategy on the satellite's propensity to call although more calls from the alpha male did cause the female to reply more frequently. We also examined the effect of relative intensity of alpha and satellite calls on the female's reply. Reduced satellite intensity increased the variance in the timing of the female response. Finally, we tested the effectiveness of the satellite's call on female phonotaxis within a two-speaker arena. Although females preferred the alpha male they were nevertheless attracted to the satellite calls regardless of the latter's relative intensity. We discuss the possible role of satellite calling as a novel conditional strategy. Copyright 2000 The Association for the Study of Animal Behaviour.

Cloning and sequence analysis of a cDNA encoding the alpha-subunit of mouse beta-N-acetylhexosaminidase and comparison with the human enzyme.

PubMed Central

Beccari, T; Hoade, J; Orlacchio, A; Stirling, J L

1992-01-01

cDNAs encoding the mouse beta-N-acetylhexosaminidase alpha-subunit were isolated from a mouse testis library. The longest of these (1.7 kb) was sequenced and showed 83% similarity with the human alpha-subunit cDNA sequence. The 5' end of the coding sequence was obtained from a genomic DNA clone. Alignment of the human and mouse sequences showed that all three putative N-glycosylation sites are conserved, but that the mouse alpha-subunit has an additional site towards the C-terminus. All eight cysteines in the human sequence are conserved in the mouse. There are an additional two cysteines in the mouse alpha-subunit signal peptide. All amino acids affected in Tay-Sachs-disease mutations are conserved in the mouse. Images Fig. 1. PMID:1379046

The distribution of alpha elements in Andromeda dwarf galaxies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vargas, Luis C.; Geha, Marla C.; Tollerud, Erik J., E-mail: luis.vargas@yale.edu

We present alpha to iron abundance ratios for 226 individual red giant branch stars in nine dwarf galaxies of the Andromeda (M31) satellite system. The abundances are measured from the combined signal of Mg, Si, Ca, and Ti lines in Keck/DEIMOS medium-resolution spectra. This constitutes the first large sample of alpha abundance ratios measured in the M31 satellite system. The dwarf galaxies in our sample exhibit a variety of alpha abundance ratios, with the average values in each galaxy ranging from approximately solar ([α/Fe] ∼ + 0.0) to alpha-enhanced ([α/Fe] ∼ + 0.5). These variations do not show a correlationmore » with internal kinematics, environment, or stellar density. We confirm radial gradients in the iron abundance of two galaxies out of the five with sufficient data (NGC 185 and And II). There is only tentative evidence for an alpha abundance radial gradient in NGC 185. We homogeneously compare our results to the Milky Way classical dwarf spheroidals, finding evidence for wider variation in average alpha abundance. In the absence of chemical abundances for the M31 stellar halo, we compare to the Milky Way stellar halo. A stellar halo comprised of disrupted M31 satellites is too metal-rich and inconsistent with the Milky Way halo alpha abundance distribution even if considering only satellites with predominantly old stellar populations. The M31 satellite population provides a second system in which to study chemical abundances of dwarf galaxies and reveals a wider variety of abundance patterns than the Milky Way.« less

Centromeric and non-centromeric satellite DNA organisation differs in holocentric Rhynchospora species.

PubMed

Ribeiro, Tiago; Marques, André; Novák, Petr; Schubert, Veit; Vanzela, André L L; Macas, Jiri; Houben, Andreas; Pedrosa-Harand, Andrea

2017-03-01

Satellite DNA repeats (or satDNA) are fast-evolving sequences usually associated with condensed heterochromatin. To test whether the chromosomal organisation of centromeric and non-centromeric satDNA differs in species with holocentric chromosomes, we identified and characterised the major satDNA families in the holocentric Cyperaceae species Rhynchospora ciliata (2n = 10), R. globosa (2n = 50) and R. tenuis (2n = 2x = 4 and 2n = 4x = 8). While conserved centromeric repeats (present in R. ciliata and R. tenuis) revealed linear signals at both chromatids, non-centromeric, species-specific satDNAs formed distinct clusters along the chromosomes. Colocalisation of both repeat types resulted in a ladder-like hybridisation pattern at mitotic chromosomes. In interphase, the centromeric satDNA was dispersed while non-centromeric satDNA clustered and partly colocalised to chromocentres. Despite the banding-like hybridisation patterns of the clustered satDNA, the identification of chromosome pairs was impaired due to the irregular hybridisation patterns of the homologues in R. tenuis and R. ciliata. These differences are probably caused by restricted or impaired meiotic recombination as reported for R. tenuis, or alternatively by complex chromosome rearrangements or unequal condensation of homologous metaphase chromosomes. Thus, holocentricity influences the chromosomal organisation leading to differences in the distribution patterns and condensation dynamics of centromeric and non-centromeric satDNA.

Alpha-fetoprotein and Fanconi Anemia: Relevance to DNA Repair and Breast Cancer Susceptibility.

PubMed

Lakhi, Nisha A; Mizejewski, Gerald J

2017-02-01

Elevations of serum alpha-fetoprotein (sAFP) have been reported in fetal and infant states of anemia. Fanconi anemia (FA) belongs to a family of genetic instability disorders which lack the capability to repair DNA breaks. The lesion occurs at a checkpoint regulatory step of the G2 to mitotic transition, allowing FA cells to override cell-cycle arrest. FA DNA repair pathways contain complementation groups known as FANC proteins. FANC proteins form multi-protein complexes with BRCA proteins and are involved in homologous DNA repair. An impaired cascade in these events imparts an increased breast cancer susceptibility to female FA patients. Elevations of sAFP have availed this fetal protein to serve as a biomarker for FA disease. However, the origin of the synthesis of sAFA has not been determined in FA patients. We hypothesize that hematopoietic multipotent progenitor stem cells in the bone marrow are the source of sAFP production in FA patients.

The circular dichroism properties of phi W-14 DNA containing alpha-putrescinylthymine.

PubMed

Spetter, S; Chen, C; Warren, R A; Hanlon, S

1985-03-08

The circular dichroism properties of phi W-14 DNA containing alpha-putrescinylthymine and its acetylated derivative have been examined in a number of aqueous solvents. Native phi W-14 DNA exhibits a B-type CD spectrum whose characteristics do not entirely conform to what would be expected for its GC content (51%). The conformationally sensitive positive band above 260 nm has a rotational strength greater than that normally found in prokaryotic DNAs of comparable GC content, such as Escherichia coli DNA. The rotational strength of this band in the spectrum of the heat-denatured form of phi W-14 DNA, however, is similar to that of heat denatured E. coli DNA. Abolition of the positive charge on the putrescine residues of native phi W-14 DNA by reaction with CH2O or by acetylation reduces the rotational strength to a level appropriate for its GC content. Increases in the electrolyte content of the solvent have the same effect, although the rotational strength of this band in phi W-14 DNA does not become comparable to that of E. coli DNA until 6-7 M LiCl. Titration to pH 10.6 in solvents of modest electrolyte content, however, fails to appreciably affect the CD spectral properties of either native phi W-14 DNA or the derivative in which half of the secondary and all of the primary amino groups have been acetylated. On the basis of these results we have concluded that the enhanced rotational strength of the positive band above 260 nm in the CD spectrum of phi W-14 DNA is due to a conformational difference caused by an ion-pair interaction of the positively charged primary amino groups of putrescine with the phosphate backbone. The CD spectral properties, however, reveal that these differences, averaged over the entire basepair population, appear to be relatively small. The average conformation, at least in dilute aqueous solutions, seems to be an unexceptional B variant with conformational properties which would be more appropriate for a DNA of higher CG content.

Inhibition of the synthesis of polyamines and DNA in activated lymphocytes by a combination of alpha-methylornithine and methylglyoxal bis(guanylhydrazone).

PubMed

Morris, D R; Jorstad, C M; Seyfried, C E

1977-09-01

The cancer chemotherapeutic drug, methylglyoxal bis(guanylhydrazone), inhibits the synthesis of spermidine and spermine, but allows continued putrescine production in small lymphocytes stimulated by concanavalin A. DNA replication in these cells is inhibited 50% while the synthesis of protein and RNA continues normally. When excess putrescine accumulation in the presence of methylglyoxal bis(guanylhydrazone) was inhibited with alpha-methylornithine, a competitive inhibitor of ornithine decarboxylase, the inhibition of DNA replication was accentuated, with still no effect on protein or RNA synthesis. No inhibition of DNA synthesis by the combination of alpha-methylornithine and methylglyoxal bis(guanylhydrazone) was observed when the inhibitors were added after accumulation of cellular polyamines. In addition, inhibition was reversed by exogenous putrescine, spermidine, or spermine. We conclude that putrescine can fulfill in part the role normally played by spermidine and spermine in DNA replication, and that blocking putrescine synthesis in the presence of methylglyoxal bis(guanylhydrazone) amplifies the polyamine requirement. The implications of this with regard to polyamine synthesis as a site of chemotherapy are discussed.

In vitro selection of single-stranded DNA molecular recognition elements against S. aureus alpha toxin and sensitive detection in human serum.

PubMed

Hong, Ka L; Battistella, Luisa; Salva, Alysia D; Williams, Ryan M; Sooter, Letha J

2015-01-27

Alpha toxin is one of the major virulence factors secreted by Staphylococcus aureus, a bacterium that is responsible for a wide variety of infections in both community and hospital settings. Due to the prevalence of S. aureus related infections and the emergence of methicillin-resistant S. aureus, rapid and accurate diagnosis of S. aureus infections is crucial in benefiting patient health outcomes. In this study, a rigorous Systematic Evolution of Ligands by Exponential Enrichment (SELEX) variant previously developed by our laboratory was utilized to select a single-stranded DNA molecular recognition element (MRE) targeting alpha toxin with high affinity and specificity. At the end of the 12-round selection, the selected MRE had an equilibrium dissociation constant (Kd) of 93.7 ± 7.0 nM. Additionally, a modified sandwich enzyme-linked immunosorbent assay (ELISA) was developed by using the selected ssDNA MRE as the toxin-capturing element and a sensitive detection of 200 nM alpha toxin in undiluted human serum samples was achieved.

Characterization of the translation products of the major mRNA species from rabbit lactating mammary glands and construction of bacterial recombinants containing casein and alpha-lactalbumin complementary DNA.

PubMed Central

Suard, Y M; Tosi, M; Kraehenbuhl, J P

1982-01-01

Total cytoplasmic polyadenylated RNA from lactating rabbit mammary glands was analysed on methylmercury hydroxide-agarose gels. The size of the most abundant mRNA species ranged between 0.5 and 5.0 kb (kilobases), with major bands at 0.55, 0.84, 0.92, 1.18 and 2.4 kb and discrete minor bands of 1.5, 1.7, 3.0 and 3.9 kb. Translation in vitro of total mRNA with [3H]leucine or [35S]methionine as precursor yielded four major bands with apparent Mr values of 16 000, 25 000, 26 000 and 29 000. The four protein bands were identified by immunoprecipitation by using specific antisera as alpha-lactalbumin and x-, kappa- and alpha-caseins, respectively. Labelling with (35S]cysteine followed by immunoprecipitation with anti-transferrin or anti-alpha-lactalbumin sera allowed the identification of two whey proteins. Translated transferrin was resolved as an 80 000-dalton band and alpha-lactalbumin appeared as a 16 000-dalton protein. A library of recombinant plasmids containing cDNA (complementary DNA) sequences representing cytoplasmic polyadenylated RNA was used to isolate clones for the major rabbit caseins and alpha-lactalbumin. A preliminary characterization of these cDNA clones was achieved by colony hybridization with enriched RNA fractions as probes. Positive clones were identified by use of hybrid-promoted translation in vitro and immunoprecipitation of the translation products. The corresponding mRNA species were further identified by hybridizing RNA blots with radioactively labelled cDNA clones. We present the restriction map of alpha-casein and kappa-casein cDNA clones. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:6123313

Cryptic MCAT enhancer regulation in fibroblasts and smooth muscle cells. Suppression of TEF-1 mediated activation by the single-stranded DNA-binding proteins, Pur alpha, Pur beta, and MSY1.

PubMed

Carlini, Leslie E; Getz, Michael J; Strauch, Arthur R; Kelm, Robert J

2002-03-08

An asymmetric polypurine-polypyrimidine cis-element located in the 5' region of the mouse vascular smooth muscle alpha-actin gene serves as a binding site for multiple proteins with specific affinity for either single- or double-stranded DNA. Here, we test the hypothesis that single-stranded DNA-binding proteins are responsible for preventing a cryptic MCAT enhancer centered within this element from cooperating with a nearby serum response factor-interacting CArG motif to trans-activate the minimal promoter in fibroblasts and smooth muscle cells. DNA binding studies revealed that the core MCAT sequence mediates binding of transcription enhancer factor-1 to the double-stranded polypurine-polypyrimidine element while flanking nucleotides account for interaction of Pur alpha and Pur beta with the purine-rich strand and MSY1 with the complementary pyrimidine-rich strand. Mutations that selectively impaired high affinity single-stranded DNA binding by fibroblast or smooth muscle cell-derived Pur alpha, Pur beta, and MSY1 in vitro, released the cryptic MCAT enhancer from repression in transfected cells. Additional experiments indicated that Pur alpha, Pur beta, and MSY1 also interact specifically, albeit weakly, with double-stranded DNA and with transcription enhancer factor-1. These results are consistent with two plausible models of cryptic MCAT enhancer regulation by Pur alpha, Pur beta, and MSY1 involving either competitive single-stranded DNA binding or masking of MCAT-bound transcription enhancer factor-1.

Characterization of Satellite DNA Sequences from the Commercially Important Marine Rotifers Brachionus rotundiformis and Brachionus plicatilis.

PubMed

Boehm; Gibson; Lubzens

2000-01-01

This study was initiated to search for species-specific and strain-specific satellite DNA sequences for which oligonucleotide primers could be designed to differentiate between various commercially important strains of the marine monogonont rotifers Brachionus rotundiformis and Brachionus plicatilis. Two unrelated, highly reiterated satellite sequences were cloned and characterized. The eight sequenced monomers from B. rotundiformis and six from B. plicatilis had low intrarepeat variability and were similar in their overall lengths, A + T compositions, and high degrees of repeated motif substructure. However, hybridizations to 19 representative strains, sequence characterizations, and GenBank searches indicated that these two satellites are morphotype-specific and population-specific, respectively, and share little homology to each other or to other characterized sequences in the database. Primer pairs designed for the B. rotundiformis satellite confirmed hybridization specificities on polymerase chain reaction and could serve as a useful molecular diagnostic tool to identify strains belonging to the SS morphotype, which are gaining widespread usage as first feeds for marine fish in commercial production.

Molecular cloning and characterization of Hymenolepis diminuta alpha-tubulin gene.

PubMed

Mohajer-Maghari, Behrokh; Amini-Bavil-Olyaee, Samad; Webb, Rodney A; Coe, Imogen R

2007-02-01

To isolate a full-length alpha-tubulin cDNA from an eucestode, Hymenolepis diminuta, a lambda phage cDNA library was constructed. The alpha-tubulin gene was cloned, sequenced and characterized. The H. diminuta alpha-tubulin consisted of 450 amino acids. This protein contained putative sites for all posttranslational modifications as detyrosination/tyrosination at the carboxyl-terminal of protien, phosphorylation at residues R79 and K336, glycylation/glutamylation at residue G445 and acetylation at residue K40. Comparisons of H. diminuta alpha-tubulin with all full-length alpha-tubulin proteins revealed that H. diminuta alpha-tubulin possesses 10 distinctive residues, which are not found in any other alpha-tubulins. Phylogenetic analysis showed that H. diminuta alpha-tubulin has grouped in a separated branch adjacent eucestode and trematodes branch with 92% bootstrap value (1000 replicates). In conclusion, this is the first report of H. diminuta cDNA library construction, cloning and characterization of H. diminuta alpha-tubulin gene.

Impaired coactivator activity of the Gly{sub 482} variant of peroxisome proliferator-activated receptor {gamma} coactivator-1{alpha} (PGC-1{alpha}) on mitochondrial transcription factor A (Tfam) promoter

DOE Office of Scientific and Technical Information (OSTI.GOV)

Choi, Yon-Sik; Hong, Jung-Man; Lim, Sunny

2006-06-09

Mitochondrial dysfunction may cause diabetes or insulin resistance. Peroxisome proliferation-activated receptor-{gamma} (PPAR-{gamma}) coactivator-1 {alpha} (PGC-1{alpha}) increases mitochondrial transcription factor A (Tfam) resulting in mitochondrial DNA content increase. An association between a single nucleotide polymorphism (SNP), G1444A(Gly482Ser), of PGC-1{alpha} coding region and insulin resistance has been reported in some ethnic groups. In this study, we investigated whether a change of glycine to serine at codon 482 of PGC-1{alpha} affected the Tfam promoter activity. The cDNA of PGC-1{alpha} variant bearing either glycine or serine at 482 codon was transfected into Chang human hepatocyte cells. The PGC-1{alpha} protein bearing glycine had impaired coactivatormore » activity on Tfam promoter-mediated luciferase. We analyzed the PGC-1{alpha} genotype G1444A and mitochondrial DNA (mtDNA) copy number from 229 Korean leukocyte genomic DNAs. Subjects with Gly/Gly had a 20% lower amount of peripheral blood mtDNA than did subjects with Gly/Ser and Ser/Ser (p < 0.05). No correlation was observed between diabetic parameters and PGC-1{alpha} genotypes in Koreans. These results suggest that PGC-1{alpha} variants with Gly/Gly at 482nd amino acid may impair the Tfam transcription, a regulatory function of mitochondrial biogenesis, resulting in dysfunctional mtDNA replication.« less

A discriminatory function for prediction of protein-DNA interactions based on alpha shape modeling.

PubMed

Zhou, Weiqiang; Yan, Hong

2010-10-15

Protein-DNA interaction has significant importance in many biological processes. However, the underlying principle of the molecular recognition process is still largely unknown. As more high-resolution 3D structures of protein-DNA complex are becoming available, the surface characteristics of the complex become an important research topic. In our work, we apply an alpha shape model to represent the surface structure of the protein-DNA complex and developed an interface-atom curvature-dependent conditional probability discriminatory function for the prediction of protein-DNA interaction. The interface-atom curvature-dependent formalism captures atomic interaction details better than the atomic distance-based method. The proposed method provides good performance in discriminating the native structures from the docking decoy sets, and outperforms the distance-dependent formalism in terms of the z-score. Computer experiment results show that the curvature-dependent formalism with the optimal parameters can achieve a native z-score of -8.17 in discriminating the native structure from the highest surface-complementarity scored decoy set and a native z-score of -7.38 in discriminating the native structure from the lowest RMSD decoy set. The interface-atom curvature-dependent formalism can also be used to predict apo version of DNA-binding proteins. These results suggest that the interface-atom curvature-dependent formalism has a good prediction capability for protein-DNA interactions. The code and data sets are available for download on http://www.hy8.com/bioinformatics.htm kenandzhou@hotmail.com.

Heterochromatin-Encoded Satellite RNAs Induce Breast Cancer.

PubMed

Zhu, Quan; Hoong, Nien; Aslanian, Aaron; Hara, Toshiro; Benner, Christopher; Heinz, Sven; Miga, Karen H; Ke, Eugene; Verma, Sachin; Soroczynski, Jan; Yates, John R; Hunter, Tony; Verma, Inder M

2018-06-07

Heterochromatic repetitive satellite RNAs are extensively transcribed in a variety of human cancers, including BRCA1 mutant breast cancer. Aberrant expression of satellite RNAs in cultured cells induces the DNA damage response, activates cell cycle checkpoints, and causes defects in chromosome segregation. However, the mechanism by which satellite RNA expression leads to genomic instability is not well understood. Here we provide evidence that increased levels of satellite RNAs in mammary glands induce tumor formation in mice. Using mass spectrometry, we further show that genomic instability induced by satellite RNAs occurs through interactions with BRCA1-associated protein networks required for the stabilization of DNA replication forks. Additionally, de-stabilized replication forks likely promote the formation of RNA-DNA hybrids in cells expressing satellite RNAs. These studies lay the foundation for developing novel therapeutic strategies that block the effects of non-coding satellite RNAs in cancer cells. Copyright © 2018 Elsevier Inc. All rights reserved.

Patterns of DNA variation among three centromere satellite families in Arabidopsis halleri and A. lyrata.

PubMed

Kawabe, Akira; Charlesworth, Deborah

2007-02-01

We describe patterns of DNA variation among the three centromeric satellite families in Arabidopsis halleri and lyrata. The newly studied subspecies (A. halleri ssp. halleri and A. lyrata ssp. lyrata and petraea), like the previously studied A. halleri ssp. gemmifera and A. lyrata ssp. kawasakiana, have three different centromeric satellite families, the older pAa family (also present in A. arenosa) and two families, pAge1 and pAge2, that probably evolved more recently. Sequence variability is high in all three satellite families, and the pAa sequences do not cluster by their species of origin. Diversity in the pAge2 family is complex, and different from variation among copies of the other two families, showing clear evidence for exchange events among family members, especially in A. halleri ssp. halleri. In A. lyrata ssp. lyrata there is some evidence for recent rapid spread of pAge2 variants, suggesting selection favoring these sequences.

The nuclear protein PH5P of the inter-alpha-inhibitor superfamily: a missing link between poly(ADP-ribose)polymerase and the inter-alpha-inhibitor family and a novel actor of DNA repair?

PubMed

Jean, L; Risler, J L; Nagase, T; Coulouarn, C; Nomura, N; Salier, J P

1999-03-05

Poly(ADP-ribose)polymerase is a nuclear NAD-dependent enzyme and an essential nick sensor involved in cellular processes where nicking and rejoining of DNA strands are required. The inter-alpha-inhibitor family is comprized of several plasma proteins that all harbor one or more so-called heavy chains designated H1-H4. The latter originate from precursor polypeptides H1P-H4P whose upper two thirds are highly homologous. We now describe a novel protein that includes (i) a so-called BRCT domain found in many proteins involved in DNA repair, (ii) an area that is homologous to the NAD-dependent catalytic domain of poly(ADP-ribose)polymerase, (iii) an area that is homologous to the upper two thirds of precursor polypeptides H1P-H4P and (iv) a proline-rich region with a potential nuclear localization signal. This protein now designated PH5P points to as yet unsuspected links between poly(ADP-ribose)polymerase and the inter-alpha-inhibitor family and is likely to be involved in DNA repair.

Hypoxia-inducible factor 1 mediates hypoxia-induced cardiomyocyte lipid accumulation by reducing the DNA binding activity of peroxisome proliferator-activated receptor {alpha}/retinoid X receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Belanger, Adam J.; Luo Zhengyu; Vincent, Karen A.

2007-12-21

In response to cellular hypoxia, cardiomyocytes adapt to consume less oxygen by shifting ATP production from mitochondrial fatty acid {beta}-oxidation to glycolysis. The transcriptional activation of glucose transporters and glycolytic enzymes by hypoxia is mediated by hypoxia-inducible factor 1 (HIF-1). In this study, we examined whether HIF-1 was involved in the suppression of mitochondrial fatty acid {beta}-oxidation in hypoxic cardiomyocytes. We showed that either hypoxia or adenovirus-mediated expression of a constitutively stable hybrid form (HIF-1{alpha}/VP16) suppressed mitochondrial fatty acid metabolism, as indicated by an accumulation of intracellular neutral lipid. Both treatments also reduced the mRNA levels of muscle carnitine palmitoyltransferasemore » I which catalyzes the rate-limiting step in the mitochondrial import of fatty acids for {beta}-oxidation. Furthermore, adenovirus-mediated expression of HIF-1{alpha}/VP16 in cardiomyocytes under normoxic conditions also mimicked the reduction in the DNA binding activity of peroxisome proliferator-activated receptor {alpha} (PPAR{alpha})/retinoid X receptor (RXR), in the presence or absence of a PPAR{alpha} ligand. These results suggest that HIF-1 may be involved in hypoxia-induced suppression of fatty acid metabolism in cardiomyocytes by reducing the DNA binding activity of PPAR{alpha}/RXR.« less

Alpha3, a transposable element that promotes host sexual reproduction.

PubMed

Barsoum, Emad; Martinez, Paula; Aström, Stefan U

2010-01-01

Theoretical models predict that selfish DNA elements require host sex to persist in a population. Therefore, a transposon that induces sex would strongly favor its own spread. We demonstrate that a protein homologous to transposases, called alpha3, was essential for mating type switch in Kluyveromyces lactis. Mutational analysis showed that amino acids conserved among transposases were essential for its function. During switching, sequences in the 5' and 3' flanking regions of the alpha3 gene were joined, forming a DNA circle, showing that alpha3 mobilized from the genome. The sequences encompassing the alpha3 gene circle junctions in the mating type alpha (MATalpha) locus were essential for switching from MATalpha to MATa, suggesting that alpha3 mobilization was a coupled event. Switching also required a DNA-binding protein, Mating type switch 1 (Mts1), whose binding sites in MATalpha were important. Expression of Mts1 was repressed in MATa/MATalpha diploids and by nutrients, limiting switching to haploids in low-nutrient conditions. A hairpin-capped DNA double-strand break (DSB) was observed in the MATa locus in mre11 mutant strains, indicating that mating type switch was induced by MAT-specific DSBs. This study provides empirical evidence for selfish DNA promoting host sexual reproduction by mediating mating type switch.

A new stable alpha chain variant: Hb Basel [alpha14(A12)Trp-->Leu (alpha1)].

PubMed

Hergersberg, Martin; Brunner-Agten, Saskia; Kühne, Thomas; Paulussen, Michael; Huber, Andreas R

2010-06-01

We describe a heterozygosity for a new missense mutation on the alpha1-globin gene of an 18-year-old woman of Portuguese ancestry with severe hypochromic anemia and iron deficiency. Hemoglobin (Hb) analysis by high performance liquid chromatography (HPLC) found a prominent peak constituting about 12% of total Hb. Sequencing of the globin genes of the index patient found the mutation alpha14(A12)Trp-->Leu (alpha1), HBA1:c.44GDNA of the mother, which provides evidence that the variant is stable and does not have direct pathophysiological or hematological consequences.

Cloning, expression, and mapping of allergenic determinants of alphaS1-casein, a major cow's milk allergen.

PubMed

Schulmeister, Ulrike; Hochwallner, Heidrun; Swoboda, Ines; Focke-Tejkl, Margarete; Geller, Beate; Nystrand, Mats; Härlin, Annika; Thalhamer, Josef; Scheiblhofer, Sandra; Keller, Walter; Niggemann, Bodo; Quirce, Santiago; Ebner, Christoph; Mari, Adriano; Pauli, Gabrielle; Herz, Udo; Valenta, Rudolf; Spitzauer, Susanne

2009-06-01

Milk is one of the first components introduced into human diet. It also represents one of the first allergen sources, which induces IgE-mediated allergies in childhood ranging from gastrointestinal, skin, and respiratory manifestations to severe life-threatening manifestations, such as anaphylaxis. Here we isolated a cDNA coding for a major cow's milk allergen, alphaS1-casein, from a bovine mammary gland cDNA library with allergic patients' IgE Abs. Recombinant alphaS1-casein was expressed in Escherichia coli, purified, and characterized by circular dichroism as a folded protein. IgE epitopes of alphaS1-casein were determined with recombinant fragments and synthetic peptides spanning the alphaS1-casein sequence using microarrayed components and sera from 66 cow's milk-sensitized patients. The allergenic activity of ralphaS1-casein and the alphaS1-casein-derived peptides was determined using rat basophil leukemia cells transfected with human FcepsilonRI, which had been loaded with the patients' serum IgE. Our results demonstrate that ralphaS1-casein as well as alphaS1-casein-derived peptides exhibit IgE reactivity, but mainly the intact ralphaS1-casein induced strong basophil degranulation. These results suggest that primarily intact alphaS1-casein or larger IgE-reactive portions thereof are responsible for IgE-mediated symptoms of food allergy. Recombinant alphaS1-casein as well as alphaS1-casein-derived peptides may be used in clinical studies to further explore pathomechanisms of food allergy as well as for the development of new diagnostic and therapeutic strategies for milk allergy.

Resolution of the diadenosine 5',5"'-P1,P4-tetraphosphate binding subunit from a multiprotein form of HeLa cell DNA polymerase alpha.

PubMed Central

Baril, E; Bonin, P; Burstein, D; Mara, K; Zamecnik, P

1983-01-01

A diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) binding subunit has been resolved from a high molecular weight (640,000) multiprotein form of DNA polymerase alpha [deoxynucleoside triphosphate:DNA nucleotidyltransferase (DNA-directed), EC 2.7.7.7] from HeLa cells [DNA polymerase alpha 2 of Lamothe, P., Baril, B., Chi, A., Lee, L. & Baril, E. (1981) Proc. Natl. Acad. Sci. USA 78, 4723-4727]. The Ap4A binding activity copurifies with the DNA polymerizing activity during the course of purification. Hydrophobic chromatography on butylagarose resolves the Ap4A binding activity from the DNA polymerase. The Ap4A binding activity is protein in nature since the binding of Ap4A is abolished by treatment of the isolated binding activity with proteinase K but is insensitive to treatment with DNase or RNase. The molecular weight of the Ap4A binding protein, as determined by polyacrylamide gel electrophoresis under nondenaturing conditions or by NaDodSO4/polyacrylamide gel electrophoresis after photoaffinity labeling of the protein with [32P]Ap4A is 92,000 or 47,000. The binding activity of this protein is highly specific for Ap4A. Images PMID:6576366

Transcriptional regulation of human retinoic acid receptor-alpha (RAR-{alpha}) by Wilms` tumour gene product

DOE Office of Scientific and Technical Information (OSTI.GOV)

Goodyer, P.R.; Torban, E.; Dehbi, M.

1994-09-01

The Wilms` tumor gene encodes a 47-49 kDa transcription factor expressed in kidney, gonads and mesothelium during embryogenesis. Inherited mutations of WT1 lead to aberrant urogenital development and Wilms` tumor, but the role of WT1 in development is not fully understood. Since the human RAR-{alpha} gene contains a potential WT1 binding site at its 5{prime} end, we studied the effect of WT1 co-transfection on expression of an RAR-{alpha} promoter/CAT reporter construct in COS cells. COS cells were plated at 5X10{sup 5} cells/dish in DMEM with 10% FBS and transfected by the Ca/PO4 method with an expression plasmid containing the full-lengthmore » WT1 (-/-) cDNA under the control of the CMV promoter, plasmid containing the RAR-{alpha} promoter (-519 to +36)/CAT reporter and TK/growth hormone plasmid to control for efficiency of transfection. CAT/GH activity at 48 hours was inhibited by co-transfection with increasing amounts of WT1 (-/-); maximum inhibition = 5% of control. WT1 co-transfection did not affect expression of TKGH, nor of a CMV-CAT vector. Expression of WT1 protein in tranfected COS cells was demonstrated by Western blotting. Minimal inhibiton of RAR-{alpha}/CAT activity was seen when cells were co-transfected with vectors containing WT1 deletion mutants, alternate WT1 splicing variants, or WT1 (-/-) cDNA bearing a mutation identified in a patient with Drash syndrome. Gel shift assays indicated binding of WT1 to RAR-{alpha} cDNA but not to an RAR-{alpha} deletion mutant lacking the GCGGGGGGCG site. These observations suggest that WT1 may function to regulate RAR-{alpha} expression during normal development.« less

The Past, Present, and Future of Human Centromere Genomics

PubMed Central

Aldrup-MacDonald, Megan E.; Sullivan, Beth A.

2014-01-01

The centromere is the chromosomal locus essential for chromosome inheritance and genome stability. Human centromeres are located at repetitive alpha satellite DNA arrays that compose approximately 5% of the genome. Contiguous alpha satellite DNA sequence is absent from the assembled reference genome, limiting current understanding of centromere organization and function. Here, we review the progress in centromere genomics spanning the discovery of the sequence to its molecular characterization and the work done during the Human Genome Project era to elucidate alpha satellite structure and sequence variation. We discuss exciting recent advances in alpha satellite sequence assembly that have provided important insight into the abundance and complex organization of this sequence on human chromosomes. In light of these new findings, we offer perspectives for future studies of human centromere assembly and function. PMID:24683489

Modeling proton and alpha elastic scattering in liquid water in Geant4-DNA

NASA Astrophysics Data System (ADS)

Tran, H. N.; El Bitar, Z.; Champion, C.; Karamitros, M.; Bernal, M. A.; Francis, Z.; Ivantchenko, V.; Lee, S. B.; Shin, J. I.; Incerti, S.

2015-01-01

Elastic scattering of protons and alpha (α) particles by water molecules cannot be neglected at low incident energies. However, this physical process is currently not available in the "Geant4-DNA" extension of the Geant4 Monte Carlo simulation toolkit. In this work, we report on theoretical differential and integral cross sections of the elastic scattering process for 100 eV-1 MeV incident protons and for 100 eV-10 MeV incident α particles in liquid water. The calculations are performed within the classical framework described by Everhart et al., Ziegler et al. and by the ICRU 49 Report. Then, we propose an implementation of the corresponding classes into the Geant4-DNA toolkit for modeling the elastic scattering of protons and α particles. Stopping powers as well as ranges are also reported. Then, it clearly appears that the account of the elastic scattering process in the slowing-down of the charged particle improves the agreement with the existing data in particular with the ICRU recommendations.

Complementary DNA sequences encoding the multimammate rat MHC class II DQ alpha and beta chains and cross-species sequence comparison in rodents.

PubMed

de Bellocq, J Goüy; Leirs, H

2009-09-01

Sequences of the complete open reading frame (ORF) for rodents major histocompatibility complex (MHC) class II genes are rare. Multimammate rat (Mastomys natalensis) complementary DNA (cDNA) encoding the alpha and beta chains of MHC class II DQ gene was cloned from a rapid amplifications of cDNA Emds (RACE) cDNA library. The ORFs consist of 801 and 771 bp encoding 266 and 256 amino acid residues for DQB and DQA, respectively. The genomic structure of Mana-DQ genes is globally analogous to that described for other rodents except for the insertion of a serine residue in the signal peptide of Mana-DQB, which is unique among known rodents.

Z-DNA binding protein from chicken blood nuclei

NASA Technical Reports Server (NTRS)

Herbert, A. G.; Spitzner, J. R.; Lowenhaupt, K.; Rich, A.

1993-01-01

A protein (Z alpha) that appears to be highly specific for the left-handed Z-DNA conformer has been identified in chicken blood nuclear extracts. Z alpha activity is measured in a band-shift assay by using a radioactive probe consisting of a (dC-dG)35 oligomer that has 50% of the deoxycytosines replaced with 5-bromodeoxycytosine. In the presence of 10 mM Mg2+, the probe converts to the Z-DNA conformation and is bound by Z alpha. The binding of Z alpha to the radioactive probe is specifically blocked by competition with linear poly(dC-dG) stabilized in the Z-DNA form by chemical bromination but not by B-form poly(dC-dG) or boiled salmon-sperm DNA. In addition, the binding activity of Z alpha is competitively blocked by supercoiled plasmids containing a Z-DNA insert but not by either the linearized plasmid or by an equivalent amount of the parental supercoiled plasmid without the Z-DNA-forming insert. Z alpha can be crosslinked to the 32P-labeled brominated probe with UV light, allowing us to estimate that the minimal molecular mass of Z alpha is 39 kDa.

Detection of minor and major satellite DNA in cytokinesis-blocked mouse splenocytes by a PRINS tandem labelling approach.

PubMed

Russo, A; Tommasi, A M; Renzi, L

1996-11-01

A protocol for the simultaneous visualization of minor and major satellite DNA by primed in situ DNA synthesis (PRINS) was developed in cytokinesis-blocked murine splenocytes. After individuation of optimal experimental conditions, a micronucleus (MN) test was carried out by treating splenocytes in vitro with the clastogenic agent mitomycin C and the aneugenic compound Colcemid. It was found that PRINS gives highly reproducible results, also comparable with the literature on MN results obtained by fluorescent in situ hybridization (FISH). Therefore the PRINS methodology may be proposed as a fast alternative to FISH for the characterization of induced MN.

Properties of some monkey DNA sequences obtained by a procedure that enriches for DNA replication origins.

PubMed

Zannis-Hadjopoulos, M; Kaufmann, G; Wang, S S; Lechner, R L; Karawya, E; Hesse, J; Martin, R G

1985-07-01

Twelve clones of monkey DNA obtained by a procedure that enriches 10(3)- to 10(4)-fold for nascent sequences activated early in S phase (G. Kaufmann, M. Zannis-Hadjopoulos, and R. G. Martin, Mol. Cell. Biol. 5:721-727, 1985) have been examined. Only 2 of the 12 ors sequences (origin-enriched sequences) are unique (ors1 and ors8). Three contain the highly reiterated Alu family (ors3, ors9, and ors11). One contains the highly reiterated alpha-satellite family (ors12), but none contain the Kpn family. Those remaining contain middle repetitive sequences. Two examples of the same middle repetitive sequence were found (ors2 and ors6). Three of the middle repetitive sequences (the ors2-ors6 pair, ors5, and ors10) are moderately dispersed; one (ors4) is highly dispersed. The last, ors7, has been mapped to the bona fide replication origin of the D loop of mitochondrial DNA. Of the nine ors sequences tested, half possess snapback (intrachain reannealing) properties.

Effect of human alpha 2HS glycoprotein on mouse macrophage function.

PubMed Central

Lewis, J G; André, C M

1980-01-01

alpha 2HS glycoprotein was isolated from normal adult serum. The ability of alpha 2HS glycoprotein to promote the endocytosis of radiolabelled DNA and radiolabelled latex particles by mouse macrophages was investigated. The results using both radiolabelled latex particles and radiolabelled DNA show that alpha 2HS glycoprotein enhances the ability of mouse macrophages to take up these radiolabelled substrates as compared to control cells. Images Figure 1 Figure 2 PMID:7439929

Birth, evolution, and transmission of satellite-free mammalian centromeric domains.

PubMed

Nergadze, Solomon G; Piras, Francesca M; Gamba, Riccardo; Corbo, Marco; Cerutti, Federico; McCarter, Joseph G W; Cappelletti, Eleonora; Gozzo, Francesco; Harman, Rebecca M; Antczak, Douglas F; Miller, Donald; Scharfe, Maren; Pavesi, Giulio; Raimondi, Elena; Sullivan, Kevin F; Giulotto, Elena

2018-06-01

Mammalian centromeres are associated with highly repetitive DNA (satellite DNA), which has so far hindered molecular analysis of this chromatin domain. Centromeres are epigenetically specified, and binding of the CENPA protein is their main determinant. In previous work, we described the first example of a natural satellite-free centromere on Equus caballus Chromosome 11. Here, we investigated the satellite-free centromeres of Equus asinus by using ChIP-seq with anti-CENPA antibodies. We identified an extraordinarily high number of centromeres lacking satellite DNA (16 of 31). All of them lay in LINE- and AT-rich regions. A subset of these centromeres is associated with DNA amplification. The location of CENPA binding domains can vary in different individuals, giving rise to epialleles. The analysis of epiallele transmission in hybrids (three mules and one hinny) showed that centromeric domains are inherited as Mendelian traits, but their position can slide in one generation. Conversely, centromere location is stable during mitotic propagation of cultured cells. Our results demonstrate that the presence of more than half of centromeres void of satellite DNA is compatible with genome stability and species survival. The presence of amplified DNA at some centromeres suggests that these arrays may represent an intermediate stage toward satellite DNA formation during evolution. The fact that CENPA binding domains can move within relatively restricted regions (a few hundred kilobases) suggests that the centromeric function is physically limited by epigenetic boundaries. © 2018 Nergadze et al.; Published by Cold Spring Harbor Laboratory Press.

Total alpha-globin gene cluster deletion has high frequency in Filipinos

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hunt, J.A.; Haruyama, A.Z.; Chu, B.M.

1994-09-01

Most {alpha}-thalassemias [Thal] are due to large deletions. In Southeast Asians, the (--{sup SEA}) double {alpha}-globin gene deletion is common, 3 (--{sup Tot}) total {alpha}-globin cluster deletions are known: Filipino (--{sup Fil}), Thai (--{sup Thai}), and Chinese (--{sup Chin}). In a Hawaii Thal project, provisional diagnosis of {alpha}-Thal-1 heterozygotes was based on microcytosis, normal isoelectric focusing, and no iron deficiency. One in 10 unselected Filipinos was an {alpha}-Thal-1 heterozygote, 2/3 of these had a (--{sup Tot}) deletion: a {var_sigma}-cDNA probe consistently showed fainter intensity of the constant 5.5 kb {var_sigma}{sub 2} BamHI band, with no heterzygosity for {var_sigma}-globin region polymorphisms;more » {alpha}-cDNA or {var_sigma}-cDNA probes showed no BamHI or BglII bands diagnostic of the (--{sup SEA}) deletion; bands for the (-{alpha}) {alpha}-Thal-2 single {alpha}-globin deletions were only seen in Hb H cases. A reliable monoclonal anti-{var_sigma}-peptide antibody test for the (--{sup SEA}) deletion was always negative in (--{sup Tot}) samples. Southern digests with the Lo probe, a gift from D. Higgs of Oxford Univ., confirmed that 49 of 50 (--{sup Tot}) chromosomes in Filipinos were (--{sup Fil}). Of 20 {alpha}-Thal-1 hydrops born to Filipinos, 11 were (--{sup Fil}/--{sup SEA}) compound heterozygotes; 9 were (--{sup SEA}/--{sup SEA}) homozygotes, but none was a (--{sup Fil}/--{sup Fil}).« less

Identification of alpha-enolase as a nuclear DNA-binding protein in the zona fasciculata but not the zona reticularis of the human adrenal cortex.

PubMed

Wang, Weiye; Wang, Lishan; Endoh, Akira; Hummelke, Geoffrey; Hawks, Christina L; Hornsby, Peter J

2005-01-01

In order to establish whether there are differences in DNA-binding proteins between zona fasciculata (ZF) and zona reticularis (ZR) cells of the human adrenal cortex, we prepared nuclear extracts from separated ZF and ZR cells. The formation of DNA-protein complexes was studied using an element in the first intron of the type I and type II 3beta-hydroxysteroid dehydrogenase genes (HSD3B1 and HSD3B2). Using the element in the HSD3B2 gene as a probe, a complex (C1) was formed with extracts from ZF cells but was formed only at a low level with ZR cell extracts. Another pair of complexes (C2/C3) was formed with both ZF and ZR cell extracts. The ZF-specific protein forming C1 was enriched by column chromatography on DEAE-Sepharose and carboxymethyl-Sepharose. Oligonucleotide competition analysis on the enriched fraction gave results consistent with those obtained on the unfractionated material. A further enrichment was brought about by passing the protein over an oligonucleotide affinity column based on the HSD3B2 element. The protein bound to the column was identified as alpha-enolase by mass spectrometry. Although alpha-enolase is a glycolytic enzyme, it binds to specific DNA sequences and has been found to be present in nuclei of various cell types. We performed immunohistochemistry on sections of adult human adrenal cortex and found alpha-enolase to be located in nuclei of ZF cells but to be predominantly cytoplasmic in ZR cells. Transfection of an alpha-enolase expression vector into NCI-H295R human adrenocortical cells increased HSD3B2 promoter activity, suggesting a possible functional role for this protein in regulation of HSD3B2 expression.

Identification and characterization of a subtelomeric satellite DNA in Callitrichini monkeys.

PubMed

Araújo, Naiara Pereira; de Lima, Leonardo Gomes; Dias, Guilherme Borges; Kuhn, Gustavo Campos Silva; de Melo, Alan Lane; Yonenaga-Yassuda, Yatiyo; Stanyon, Roscoe; Svartman, Marta

2017-08-01

Repetitive DNAs are abundant fast-evolving components of eukaryotic genomes, which often possess important structural and functional roles. Despite their ubiquity, repetitive DNAs are poorly studied when compared with the genic fraction of genomes. Here, we took advantage of the availability of the sequenced genome of the common marmoset Callithrix jacchus to assess its satellite DNAs (satDNAs) and their distribution in Callitrichini. After clustering analysis of all reads and comparisons by similarity, we identified a satDNA composed by 171 bp motifs, named MarmoSAT, which composes 1.09% of the C. jacchus genome. Fluorescent in situ hybridization on chromosomes of species from the genera Callithrix, Mico and Callimico showed that MarmoSAT had a subtelomeric location. In addition to the common monomeric, we found that MarmoSAT was also organized in higher-order repeats of 338 bp in Callimico goeldii. Our phylogenetic analyses showed that MarmoSAT repeats from C. jacchus lack chromosome-specific features, suggesting exchange events among subterminal regions of non-homologous chromosomes. MarmoSAT is transcribed in several tissues of C. jacchus, with the highest transcription levels in spleen, thymus and heart. The transcription profile and subtelomeric location suggest that MarmoSAT may be involved in the regulation of telomerase and modulation of telomeric chromatin. © The Author 2017. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

Sodium butyrate rescues dopaminergic cells from alpha-synuclein-induced transcriptional deregulation and DNA damage.

PubMed

Paiva, Isabel; Pinho, Raquel; Pavlou, Maria Angeliki; Hennion, Magali; Wales, Pauline; Schütz, Anna-Lena; Rajput, Ashish; Szego, Éva M; Kerimoglu, Cemil; Gerhardt, Ellen; Rego, Ana Cristina; Fischer, André; Bonn, Stefan; Outeiro, Tiago F

2017-06-15

Alpha-synuclein (aSyn) is considered a major culprit in Parkinson's disease (PD) pathophysiology. However, the precise molecular function of the protein remains elusive. Recent evidence suggests that aSyn may play a role on transcription regulation, possibly by modulating the acetylation status of histones. Our study aimed at evaluating the impact of wild-type (WT) and mutant A30P aSyn on gene expression, in a dopaminergic neuronal cell model, and decipher potential mechanisms underlying aSyn-mediated transcriptional deregulation. We performed gene expression analysis using RNA-sequencing in Lund Human Mesencephalic (LUHMES) cells expressing endogenous (control) or increased levels of WT or A30P aSyn. Compared to control cells, cells expressing both aSyn variants exhibited robust changes in the expression of several genes, including downregulation of major genes involved in DNA repair. WT aSyn, unlike A30P aSyn, promoted DNA damage and increased levels of phosphorylated p53. In dopaminergic neuronal cells, increased aSyn expression led to reduced levels of acetylated histone 3. Importantly, treatment with sodium butyrate, a histone deacetylase inhibitor (HDACi), rescued WT aSyn-induced DNA damage, possibly via upregulation of genes involved in DNA repair. Overall, our findings provide novel and compelling insight into the mechanisms associated with aSyn neurotoxicity in dopaminergic cells, which could be ameliorated with an HDACi. Future studies will be crucial to further validate these findings and to define novel possible targets for intervention in PD. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

A new gene deletion in the alpha-like globin gene cluster as the molecular basis for the rare alpha-thalassemia-1(--/alpha alpha) in blacks: HbH disease in sickle cell trait.

PubMed

Steinberg, M H; Coleman, M B; Adams, J G; Hartmann, R C; Saba, H; Anagnou, N P

1986-02-01

A novel deletion of at least 26 kilobase of DNA, including both alpha-globin genes, the psi alpha- and psi zeta-globin genes, but sparing the functional zeta-gene was found in a 10-year-old black boy with HbH disease and sickle cell trait. This particular deletion has not previously been described in blacks. Its existence makes it likely that the absence of Hb Barts hydrops fetalis in blacks is due to the rarity of the chromosome lacking two alpha-globin genes rather than a result of early embryonic death due to the failure to synthesize embryonic hemoglobins because of deletion of functional zeta-globin genes.

Folate receptor {alpha} regulates cell proliferation in mouse gonadotroph {alpha}T3-1 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yao, Congjun; Evans, Chheng-Orn; Stevens, Victoria L.

We have previously found that the mRNA and protein levels of the folate receptor alpha (FR{alpha}) are uniquely over-expressed in clinically human nonfunctional (NF) pituitary adenomas, but the mechanistic role of FR{alpha} has not fully been determined. We investigated the effect of FR{alpha} over-expression in the mouse gonadotroph {alpha}T3-1 cell line as a model for NF pituitary adenomas. We found that the expression and function of FR{alpha} were strongly up-regulated, by Western blotting and folic acid binding assay. Furthermore, we found a higher cell growth rate, an enhanced percentage of cells in S-phase by BrdU assay, and a higher PCNAmore » staining. These observations indicate that over-expression of FR{alpha} promotes cell proliferation. These effects were abrogated in the same {alpha}T3-1 cells when transfected with a mutant FR{alpha} cDNA that confers a dominant-negative phenotype by inhibiting folic acid binding. Finally, by real-time quantitative PCR, we found that mRNA expression of NOTCH3 was up-regulated in FR{alpha} over-expressing cells. In summary, our data suggests that FR{alpha} regulates pituitary tumor cell proliferation and mechanistically may involve the NOTCH pathway. Potentially, this finding could be exploited to develop new, innovative molecular targeted treatment for human NF pituitary adenomas.« less

Combination alpha-interferon and lamivudine therapy for alpha-interferon-resistant chronic hepatitis B infection: results of a pilot study.

PubMed

Mutimer, D; Naoumov, N; Honkoop, P; Marinos, G; Ahmed, M; de Man, R; McPhillips, P; Johnson, M; Williams, R; Elias, E; Schalm, S

1998-06-01

Alpha-interferon achieves seroconversion in about one third of naive patients. Attempts to achieve seroconversion in patients who have previously failed alpha-interferon have proved disappointing. Combination chemotherapy (alpha-interferon with a nucleoside analogue) might provide a treatment alternative for these patients. We have undertaken a phase 2 study in 20 patients who had previously failed at least one course of alpha-interferon. The study was designed to assess the safety, tolerability and efficacy of the combination. All patients were treated for 16 weeks with alpha-interferon in combination with 12 or 16 weeks of Lamivudine (3'TC). Patients were followed for 16 weeks post-treatment. Pharmacokinetic studies were performed to identify/exclude significant pharmacokinetic drug interaction. The combination was well tolerated, and side-effects of the combination were indistinguishable from the recognised side-effects of alpha-interferon. Pharmacokinetic studies performed on days 1 and 29 did not show any significant interaction. All patients achieved HBV DNA clearance during treatment, but 19 relapsed at the end of treatment. HBeAg/anti-HBe seroconversion was observed for four patients, but was sustained for a single patient (who also had sustained DNA clearance). Combination therapy with alpha-interferon and lamivudine given for 16 weeks appears safe and is well tolerated. However, for this group of patients who had previously failed interferon monotherapy, the efficacy of combination interferon/lamivudine therapy appears disappointing, and other treatment strategies should be investigated.

Structural basis of nucleic-acid recognition and double-strand unwinding by the essential neuronal protein Pur-alpha

PubMed Central

Weber, Janine; Bao, Han; Hartlmüller, Christoph; Wang, Zhiqin; Windhager, Almut; Janowski, Robert; Madl, Tobias; Jin, Peng; Niessing, Dierk

2016-01-01

The neuronal DNA-/RNA-binding protein Pur-alpha is a transcription regulator and core factor for mRNA localization. Pur-alpha-deficient mice die after birth with pleiotropic neuronal defects. Here, we report the crystal structure of the DNA-/RNA-binding domain of Pur-alpha in complex with ssDNA. It reveals base-specific recognition and offers a molecular explanation for the effect of point mutations in the 5q31.3 microdeletion syndrome. Consistent with the crystal structure, biochemical and NMR data indicate that Pur-alpha binds DNA and RNA in the same way, suggesting binding modes for tri- and hexanucleotide-repeat RNAs in two neurodegenerative RNAopathies. Additionally, structure-based in vitro experiments resolved the molecular mechanism of Pur-alpha's unwindase activity. Complementing in vivo analyses in Drosophila demonstrated the importance of a highly conserved phenylalanine for Pur-alpha's unwinding and neuroprotective function. By uncovering the molecular mechanisms of nucleic-acid binding, this study contributes to understanding the cellular role of Pur-alpha and its implications in neurodegenerative diseases. DOI: http://dx.doi.org/10.7554/eLife.11297.001 PMID:26744780

Evidence for a temperature rise in the outer layers of alpha Lyrae, from Copernicus observations of Lyman-alpha

NASA Technical Reports Server (NTRS)

Praderie, F.; Simonneau, E.; Snow, T. P., Jr.

1975-01-01

Copernicus satellite observations of the Ly-alpha profiles in alpha Lyrae (Vega) are used to determine whether classical radiative-equilibrium LTE model atmospheres can fit the thermal structure in the outer layers of that star. Two plane-parallel LTE model photospheres of alpha Lyrae are considered: a line-blanketed radiative-equilibrium model with an effective temperature of 9650 K and log g of 4.05, and the same model with a temperature of 9500 K and log g of 4.0. The profiles of the Ly-alpha wings are computed, and it is found that classical LTE models are unable to predict either the observed violet wing or the red wing longwards of 1239 A, regardless of the line source function. It is concluded that the electron temperature must increase outwards over the surface value reached in radiative equilibrium.

A new begomovirus associated with alpha- and betasatellite molecules isolated from Vernonia cinerea in China.

PubMed

Zulfiqar, Awais; Zhang, Jie; Cui, Xiaofeng; Qian, Yajuan; Zhou, Xueping; Xie, Yan

2012-01-01

A begomovirus disease complex associated with Vernonia cinerea showing yellow vein symptoms was studied. The full-length genomic DNA was comprised of 2739 nucleotides (nt) and contained the typical genome structure of begomoviruses. Comparison analysis showed that it shared the highest (78.9%) nucleotide sequence identity with recently characterized Vernonia yellow vein virus (VeYVV) from India. For associated satellites, betasatellite showed the highest nucleotide sequence identity (52.1%) with Vernonia yellow vein virus betasatellite (VeYVVB) and alphasatellite shared the highest sequence identity (70.7%) with Gossypium mustelinium symptomless alphasatellite (GMusSLA). It is a member of a distinct species with cognate alpha- and betasatellites for which the name Vernonia yellow vein Fujian virus (VeYVFjV) is proposed.

The Trypanosoma cruzi Satellite DNA OligoC-TesT and Trypanosoma cruzi Kinetoplast DNA OligoC-TesT for Diagnosis of Chagas Disease: A Multi-cohort Comparative Evaluation Study

PubMed Central

De Winne, Koen; Büscher, Philippe; Luquetti, Alejandro O.; Tavares, Suelene B. N.; Oliveira, Rodrigo A.; Solari, Aldo; Zulantay, Ines; Apt, Werner; Diosque, Patricio; Monje Rumi, Mercedes; Gironès, Nuria; Fresno, Manuel; Lopez-Velez, Rogelio; Perez-Molina, José A.; Monge-Maillo, Begoña; Garcia, Lineth; Deborggraeve, Stijn

2014-01-01

Background The Trypanosoma cruzi satellite DNA (satDNA) OligoC-TesT is a standardised PCR format for diagnosis of Chagas disease. The sensitivity of the test is lower for discrete typing unit (DTU) TcI than for TcII-VI and the test has not been evaluated in chronic Chagas disease patients. Methodology/Principal Findings We developed a new prototype of the OligoC-TesT based on kinetoplast DNA (kDNA) detection. We evaluated the satDNA and kDNA OligoC-TesTs in a multi-cohort study with 187 chronic Chagas patients and 88 healthy endemic controls recruited in Argentina, Chile and Spain and 26 diseased non-endemic controls from D.R. Congo and Sudan. All specimens were tested in duplicate. The overall specificity in the controls was 99.1% (95% CI 95.2%–99.8%) for the satDNA OligoC-TesT and 97.4% (95% CI 92.6%–99.1%) for the kDNA OligoC-TesT. The overall sensitivity in the patients was 67.9% (95% CI 60.9%–74.2%) for the satDNA OligoC-TesT and 79.1% (95% CI 72.8%–84.4%) for the kDNA OligoC-Test. Conclusions/Significance Specificities of the two T. cruzi OligoC-TesT prototypes are high on non-endemic and endemic controls. Sensitivities are moderate but significantly (p = 0.0004) higher for the kDNA OligoC-TesT compared to the satDNA OligoC-TesT. PMID:24392177

Mapping Simple Repeated DNA Sequences in Heterochromatin of Drosophila Melanogaster

PubMed Central

Lohe, A. R.; Hilliker, A. J.; Roberts, P. A.

1993-01-01

Heterochromatin in Drosophila has unusual genetic, cytological and molecular properties. Highly repeated DNA sequences (satellites) are the principal component of heterochromatin. Using probes from cloned satellites, we have constructed a chromosome map of 10 highly repeated, simple DNA sequences in heterochromatin of mitotic chromosomes of Drosophila melanogaster. Despite extensive sequence homology among some satellites, chromosomal locations could be distinguished by stringent in situ hybridizations for each satellite. Only two of the localizations previously determined using gradient-purified bulk satellite probes are correct. Eight new satellite localizations are presented, providing a megabase-level chromosome map of one-quarter of the genome. Five major satellites each exhibit a multichromosome distribution, and five minor satellites hybridize to single sites on the Y chromosome. Satellites closely related in sequence are often located near one another on the same chromosome. About 80% of Y chromosome DNA is composed of nine simple repeated sequences, in particular (AAGAC)(n) (8 Mb), (AAGAG)(n) (7 Mb) and (AATAT)(n) (6 Mb). Similarly, more than 70% of the DNA in chromosome 2 heterochromatin is composed of five simple repeated sequences. We have also generated a high resolution map of satellites in chromosome 2 heterochromatin, using a series of translocation chromosomes whose breakpoints in heterochromatin were ordered by N-banding. Finally, staining and banding patterns of heterochromatic regions are correlated with the locations of specific repeated DNA sequences. The basis for the cytochemical heterogeneity in banding appears to depend exclusively on the different satellite DNAs present in heterochromatin. PMID:8375654

DNA methylation analysis on satellite I region in blastocysts obtained from somatic cell cloned cattle.

PubMed

Yamanaka, Ken-Ichi; Kaneda, Masahiro; Inaba, Yasushi; Saito, Koji; Kubota, Kaiyu; Sakatani, Miki; Sugimura, Satoshi; Imai, Kei; Watanabe, Shinya; Takahashi, Masashi

2011-08-01

Many observations have been made on cloned embryos and on adult clones by somatic cell nuclear transfer (SCNT), but it is still unclear whether the progeny of cloned animals is presenting normal epigenetic status. Here, in order to accumulate the information for evaluating the normality of cloned cattle, we analyzed the DNA methylation status on satellite I region in blastocysts obtained from cloned cattle. Embryos were produced by artificial insemination (AI) to non-cloned or cloned dams using semen from non-cloned or cloned sires. After 7 days of AI, embryos at blastocyst stage were collected by uterine flushing. The DNA methylation levels in embryos obtained by using semen and/or oocytes from cloned cattle were similar to those in in vivo embryos from non-cloned cattle. In contrast, the DNA methylation levels in SCNT embryos were significantly higher (P < 0.01) than those in in vivo embryos from non-cloned and cloned cattle, approximately similar to those in somatic cells used as donor cells. Thus, this study provides useful information that epigenetic status may be normal in the progeny of cloned cattle, suggesting the normality of germline cells in cloned cattle. 2011 The Authors. Animal Science Journal © 2011 Japanese Society of Animal Science.

A search for Lyman-alpha emission in beta Lyrae from Copernicus

NASA Technical Reports Server (NTRS)

Kondo, Y.; Mccluskey, G. E., Jr.

1974-01-01

High-resolution (0.2 A) spectrophotometric observations of the complex eclipsing binary beta Lyrae were obtained with the Princeton Telescope Spectrometer on the Copernicus satellite. We discuss the search for L-alpha emission in beta Lyrae and compare the Copernicus results with the OAO-2 observations of the same binary system. The possible L-alpha emission features observed from OAO-2 are identified as blends of the emission lines of other elements in the vicinity of L-alpha.

[Transgenerational transmission of bovine satellite DNA in transgenic mice].

PubMed

Slominskaia, N A; Suchkova, I O; Klinskaia, T A; Zabezhinskaia, M A; Patkin, E L

2006-01-01

Genetical, cytogenetical and molecular analysis was made for 5 generations of mice transgenic for bovine satellite DNA (Sat). In all cases transgenic mice were generated by crosses of transgenic males and females with normal (CBA x C57B1) mice. No abnormalities in the founder development were noticed. A normal (near 50 %) ratio of transgenic and nontransgenic offsprings was observed in blastocysts. However, profound differences occurred in the rate of transgene bearing offsprings, depending on the sex of grandparents rather than of parents. The grandfather Sat transmission resulted in the appearance of 0-52.4 % transgenic grandchildren, whereas the grandmother transmission ended in the theoretically expected rate. This means that stabilization of transsatellite took place upon the female germ line transmission (a positive grandmother effect). It is essential that in hemizygous transsatellite mice Sat integration led to the occurrence of mammary tumors, inflammation of uterine horns, and infringement of mother care of transgenic females. Simultaneous FISH and G-banding showed Sat to be localized in the internal region of chromosome 12 near Pax 9 and Brms 11 genes. Commonly, these genes are implicated in tumorigenesis as their expression decreases. Thus, a kind of silencing effect of these genes' expression may be supposed.

Structural changes induced by binding of the high-mobility group I protein to a mouse satellite DNA sequence.

PubMed Central

Slama-Schwok, A; Zakrzewska, K; Léger, G; Leroux, Y; Takahashi, M; Käs, E; Debey, P

2000-01-01

Using spectroscopic methods, we have studied the structural changes induced in both protein and DNA upon binding of the High-Mobility Group I (HMG-I) protein to a 21-bp sequence derived from mouse satellite DNA. We show that these structural changes depend on the stoichiometry of the protein/DNA complexes formed, as determined by Job plots derived from experiments using pyrene-labeled duplexes. Circular dichroism and melting temperature experiments extended in the far ultraviolet range show that while native HMG-I is mainly random coiled in solution, it adopts a beta-turn conformation upon forming a 1:1 complex in which the protein first binds to one of two dA.dT stretches present in the duplex. HMG-I structure in the 1:1 complex is dependent on the sequence of its DNA target. A 3:1 HMG-I/DNA complex can also form and is characterized by a small increase in the DNA natural bend and/or compaction coupled to a change in the protein conformation, as determined from fluorescence resonance energy transfer (FRET) experiments. In addition, a peptide corresponding to an extended DNA-binding domain of HMG-I induces an ordered condensation of DNA duplexes. Based on the constraints derived from pyrene excimer measurements, we present a model of these nucleated structures. Our results illustrate an extreme case of protein structure induced by DNA conformation that may bear on the evolutionary conservation of the DNA-binding motifs of HMG-I. We discuss the functional relevance of the structural flexibility of HMG-I associated with the nature of its DNA targets and the implications of the binding stoichiometry for several aspects of chromatin structure and gene regulation. PMID:10777751

The ultraviolet spectra of Alpha Aquilae and Alpha Canis Minoris

NASA Technical Reports Server (NTRS)

Morton, D. C.; Bruzual A., G.; Kurucz, R. L.; Spinrad, H.

1977-01-01

Scans of Alpha Aql (A7 IV, V) and Alpha CMi (F5 IV-V) obtained with the Copernicus satellite spectrometer over the wavelength range from 2100 to 3200 A are presented along with a spectrum of the integrated solar disk over the same range procured during a calibrated rocket flight. About 1500 fairly strong absorption lines in the Alpha CMi spectrum between 2400 and 2961 A are identified by comparison with a solar atlas and by using a theoretical spectrum synthesized from a blanketed LTE model with an effective temperature of 6500 K and a surface gravity of 10,000 cm/sec per sec. The Mg II resonance doublet at 2795.528 and 2802.704 A is found to be present in all three stars together with a discontinuity at 2635 A due to Fe II, Fe I, Cr I, and Mn II. It is concluded that the Mg II resonance lines and the 2635-A continuum break would be the best spectral features for estimating the redshift of a galaxy observed at low resolution provided the redshift is not less than about 0.75.

Immunogenicity of a DNA-launched replicon-based canine parvovirus DNA vaccine expressing VP2 antigen in dogs.

PubMed

Dahiya, Shyam S; Saini, Mohini; Kumar, Pankaj; Gupta, Praveen K

2012-10-01

A replicon-based DNA vaccine encoding VP2 gene of canine parvovirus (CPV) was developed by cloning CPV-VP2 gene into a replicon-based DNA vaccine vector (pAlpha). The characteristics of a replicon-based DNA vaccine like, self-amplification of transcripts and induction of apoptosis were analyzed in transfected mammalian cells. When the pAlpha-CPV-VP2 was injected intradermal as DNA-launched replicon-based DNA vaccine in dogs, it induced CPV-specific humoral and cell mediated immune responses. The virus neutralization antibody and lymphocyte proliferative responses were higher than conventional CPV DNA vaccine and commercial CPV vaccine. These results indicated that DNA-launched replicon-based CPV DNA vaccine was effective in inducing both CPV-specific humoral and cellular immune responses and can be considered as effective alternative to conventional CPV DNA vaccine and commercial CPV vaccine. Crown Copyright © 2012. Published by Elsevier India Pvt Ltd. All rights reserved.

The alpha-spectrin gene is on chromosome 1 in mouse and man.

PubMed Central

Huebner, K; Palumbo, A P; Isobe, M; Kozak, C A; Monaco, S; Rovera, G; Croce, C M; Curtis, P J

1985-01-01

By using alpha-spectrin cDNA clones of murine and human origin and somatic cell hybrids segregating either mouse or human chromosomes, the gene for alpha-spectrin has been mapped to chromosome 1 in both species. This assignment of the mouse alpha-spectrin gene to mouse chromosome 1 by DNA hybridization strengthens the previous identification of the alpha-spectrin locus in mouse with the sph locus, which previously was mapped by linkage analysis to mouse chromosome 1, distal to the Pep-3 locus. By in situ hybridization to human metaphase chromosomes, the human alpha-spectrin gene has been localized to 1q22-1q25; interestingly, the locus for a non-Rh-linked form of elliptocytosis has been provisionally mapped to band 1q2 by family linkage studies. Images PMID:2987946

The alpha-spectrin gene is on chromosome 1 in mouse and man.

PubMed

Huebner, K; Palumbo, A P; Isobe, M; Kozak, C A; Monaco, S; Rovera, G; Croce, C M; Curtis, P J

1985-06-01

By using alpha-spectrin cDNA clones of murine and human origin and somatic cell hybrids segregating either mouse or human chromosomes, the gene for alpha-spectrin has been mapped to chromosome 1 in both species. This assignment of the mouse alpha-spectrin gene to mouse chromosome 1 by DNA hybridization strengthens the previous identification of the alpha-spectrin locus in mouse with the sph locus, which previously was mapped by linkage analysis to mouse chromosome 1, distal to the Pep-3 locus. By in situ hybridization to human metaphase chromosomes, the human alpha-spectrin gene has been localized to 1q22-1q25; interestingly, the locus for a non-Rh-linked form of elliptocytosis has been provisionally mapped to band 1q2 by family linkage studies.

Genomic structure of rat 3alpha-hydroxysteroid/dihydrodiol dehydrogenase (3alpha-HSD/DD, AKR1C9).

PubMed

Lin, H K; Hung, C F; Moore, M; Penning, T M

1999-11-01

Rat liver 3alpha-hydroxysteroid/dihydrodiol dehydrogenase (3alpha-HSD/DD) is a member of the aldo-keto reductase (AKR) superfamily. It is involved in the inactivation of steroid hormones and the metabolic activation of polycyclic aromatic hydrocarbons (PAH) by converting trans-dihydrodiols into reactive and redox-active o-quinones. The structure of the 5'-flanking region of the gene and factors involved in the constitutive and regulated expression of this gene have been reported [H.-K. Lin, T.M. Penning, Cloning, sequencing, and functional analysis of the 5'-flanking region of the rat 3alpha-hydroxysteroid/dihydrodiol dehydrogenase gene, Cancer Res. 55 (1995) 4105-4113]. We now describe the complete genomic structure of the rat type 1 3alpha-HSD/DD gene. Charon 4A and P1 genomic clones contained at least three rat genes (type 1, type 2 and type 3 3alpha-HSD/DD) each of which encoded for the same open reading frame (ORF) but differed in their exon-intron organization. 5'-RACE confirmed that the type 1 3alpha-HSD/DD gene encodes for the dominant transcript in rat liver and it was the regulation of this gene that was previously studied. The rat type 1 3alpha-HSD/DD gene is 30 kb in length and consists of nine exons and eight introns. Exon 9 encodes +931 to 966 bp of the ORF and the 1292 bp 3'-UTR implicated in mRNA stability. This genomic structure is nearly identical to the homologous human genes, type 1 3alpha-HSD (chlordecone reductase/DD4, AKR1C4), type 2 3alpha-HSD (AKR1C3) and type 3 3alpha-HSD (bile-acid binding protein, AKR1C2) genes. Three different cDNA's containing identical ORFs for 3alpha-HSD have been reported suggesting that all three genes may be expressed in rat liver. Using 5' primers corresponding to the 5'-UTR's of the three different cDNA's only one PCR fragment was obtained and corresponded to the type 1 3alpha-HSD/DD gene. These data suggested that the type 2 and type 3 3alpha-HSD/DD genes are not abundantly expressed in rat liver. It is unknown

The effect of bovine IFN-alpha on the immune response in guinea pigs vaccinated with DNA vaccine of foot-and-mouth disease virus.

PubMed

Guo, Hui-Chen; Liu, Zai-Xin; Sun, Shi-Qi; Leng, Qing-Wen; Li, Dong; Liu, Xiang-Tao; Xie, Qing-Ge

2004-10-01

In this study, we constructed recombinant plasmid pcDNA3.1/P12X3C3D including P1, 2A, 3C, 3D and part of 2B gene of FMDV and pcDNA3.1/IFN containing the gene encoding bovine IFN-alpha. We inoculated the DNA vaccine pcDNA3.1/P12X3C3D with or without pcDNA3.1/IFN to evaluate the efficiency of this DNA vaccine and the immunogenicity of DNA vaccine enhanced by the co-delivery with pcDNA3.1/IFN. After two times of vaccination with DNA vaccine, all of guinea pigs were challenged with 103 ID50 FMDV type O. Anti-FMDV antibody levels were detected by ELISA and T lymphocyte proliferation response was tested by MTT assay. The result shows that guinea pigs inoculated by pcDNA3.1/P12X3C3D alone or with pcDNA3.1/IFN generated specific antibodies and induced an FMDV-specific T lymphocyte proliferation response. FMDV challenge tests showed that one in four guinea pigs immunized by pcDNA3.1/P12X3C3D with pcDNA3.1/IFN was protected from the FMDV serotype O infection. This result indicated that the efficiency of the DNA vaccine was enhanced by co-delivery with pcDNA3.1/IFN. However, the protection rate was considerably lower than that immunized with conventional FMD vaccine.

Resolution of the discrepancy between Balmer alpha emission rates, the solar Lyman beta flux, and models of geocoronal hydrogen concentration

NASA Technical Reports Server (NTRS)

Levasseur, A.-C.; Meier, R. R.; Tinsley, B. A.

1976-01-01

New satellite Balmer alpha measurements and solar Lyman beta flux and line profile measurements, together with new measurements of the zodiacal light intensity used in correcting both ground and satellite Balmer alpha measurements for the effects of the Fraunhofer line in the zodiacal light, have been used in a reevaluation of the long-standing discrepancy between ground-based Balmer alpha emission rates and other geocoronal hydrogen parameters. The solar Lyman beta line center flux is found to be (4.1 plus or minus 1.3) billion photons per sq cm per sec per angstrom at S(10.7) equals 110 and, together with a current hydrogen model which has 92,000 atoms per cu cm at 650 km for T(inf) equals 950 K, gives good agreement between calculated Balmer alpha emission rates and the ground-based and satellite measurements.

Alpha-Lipoic Acid Downregulates IL-1β and IL-6 by DNA Hypermethylation in SK-N-BE Neuroblastoma Cells.

PubMed

Dinicola, Simona; Proietti, Sara; Cucina, Alessandra; Bizzarri, Mariano; Fuso, Andrea

2017-09-26

Alpha-lipoic acid (ALA) is a pleiotropic molecule with antioxidant and anti-inflammatory properties, of which the effects are exerted through the modulation of NF-kB. This nuclear factor, in fact, modulates different inflammatory cytokines, including IL-1b and IL-6, in different tissues and cell types. We recently showed that IL-1b and IL-6 DNA methylation is modulated in the brain of Alzheimer's disease patients, and that IL-1b expression is associated to DNA methylation in the brain of patients with tuberous sclerosis complex. These results prompted us to ask whether ALA-induced repression of IL-1b and IL-6 was dependent on DNA methylation. Therefore, we profiled DNA methylation in the 5'-flanking region of the two aforementioned genes in SK-N-BE human neuroblastoma cells cultured in presence of ALA 0.5 mM. Our experimental data pointed out that the two promoters are hypermethylated in cells supplemented with ALA, both at CpG and non-CpG sites. Moreover, the observed hypermethylation is associated with decreased mRNA expression and decreased cytokine release. These results reinforce previous findings indicating that IL-1b and IL-6 undergo DNA methylation-dependent modulation in neural models and pave the road to study the epigenetic mechanisms triggered by ALA.

De novo formed satellite DNA-based mammalian artificial chromosomes and their possible applications.

PubMed

Katona, Robert L

2015-02-01

Mammalian artificial chromosomes (MACs) are non-integrating, autonomously replicating natural chromosome-based vectors that may carry a vast amount of genetic material, which in turn enable potentially prolonged, safe, and regulated therapeutic transgene expression and render MACs as attractive genetic vectors for "gene replacement" or for controlling differentiation pathways in target cells. Satellite-DNA-based artificial chromosomes (SATACs) can be made by induced de novo chromosome formation in cells of different mammalian and plant species. These artificially generated accessory chromosomes are composed of predictable DNA sequences, and they contain defined genetic information. SATACs have already passed a number of obstacles crucial to their further development as gene therapy vectors, including large-scale purification, transfer of purified artificial chromosomes into different cells and embryos, generation of transgenic animals and germline transmission with purified SATACs, and the tissue-specific expression of a therapeutic gene from an artificial chromosome in the milk of transgenic animals. SATACs could be used in cell therapy protocols. For these methods, the most versatile target cell would be one that was pluripotent and self-renewing to address multiple disease target cell types, thus making multilineage stem cells, such as adult derived early progenitor cells and embryonic stem cells, as attractive universal host cells.

The role of DNA repair in herpesvirus pathogenesis.

PubMed

Brown, Jay C

2014-10-01

In cells latently infected with a herpesvirus, the viral DNA is present in the cell nucleus, but it is not extensively replicated or transcribed. In this suppressed state the virus DNA is vulnerable to mutagenic events that affect the host cell and have the potential to destroy the virus' genetic integrity. Despite the potential for genetic damage, however, herpesvirus sequences are well conserved after reactivation from latency. To account for this apparent paradox, I have tested the idea that host cell-encoded mechanisms of DNA repair are able to control genetic damage to latent herpesviruses. Studies were focused on homologous recombination-dependent DNA repair (HR). Methods of DNA sequence analysis were employed to scan herpesvirus genomes for DNA features able to activate HR. Analyses were carried out with a total of 39 herpesvirus DNA sequences, a group that included viruses from the alpha-, beta- and gamma-subfamilies. The results showed that all 39 genome sequences were enriched in two or more of the eight recombination-initiating features examined. The results were interpreted to indicate that HR can stabilize latent herpesvirus genomes. The results also showed, unexpectedly, that repair-initiating DNA features differed in alpha- compared to gamma-herpesviruses. Whereas inverted and tandem repeats predominated in alpha-herpesviruses, gamma-herpesviruses were enriched in short, GC-rich initiation sequences such as CCCAG and depleted in repeats. In alpha-herpesviruses, repair-initiating repeat sequences were found to be concentrated in a specific region (the S segment) of the genome while repair-initiating short sequences were distributed more uniformly in gamma-herpesviruses. The results suggest that repair pathways are activated differently in alpha- compared to gamma-herpesviruses. Copyright © 2014. Published by Elsevier Inc.

alpha-DNA II. Synthesis of unnatural alpha-anomeric oligodeoxyribonucleotides containing the four usual bases and study of their substrate activities for nucleases.

PubMed Central

Morvan, F; Rayner, B; Imbach, J L; Thenet, S; Bertrand, J R; Paoletti, J; Malvy, C; Paoletti, C

1987-01-01

This paper describes for the first time the synthesis of alpha-oligonucleotides containing the four usual bases. Two unnatural hexadeoxyribonucleotides: alpha-[d(CpApTpGpCpG)] and alpha-[d(CpGpCpApTpG)], consisting only of alpha-anomeric nucleotide units, were obtained by an improved phosphotriester method, in solution. Starting material was the four base-protected alpha-deoxyribonucleosides 3a-d. Pyrimidine alpha-deoxynucleosides 3a and 3b were prepared by self-anomerization reactions followed by selective deprotection of sugar hydroxyles, while the two purine alpha-deoxynucleosides 3c and 3d were prepared by glycosylation reactions. In the case of guanine alpha-nucleoside derivative a supplementary base-protecting group: N,N-diphenylcarbamoyl was introduced on O6-position in order to avoid side-reactions during oligonucleotide assembling. The hexadeoxynucleotide alpha-[d(CpApTpGpCpG)] was tested as substrate of selected endo- and exonucleases. In conditions where the natural corresponding beta-hexamer was completely degradated by nuclease S1 and calf spleen phosphodiesterase, the alpha-oligonucleotide remained almost intact. PMID:3575096

Evidence that steroid 5alpha-reductase isozyme genes are differentially methylated in human lymphocytes.

PubMed

Rodríguez-Dorantes, M; Lizano-Soberón, M; Camacho-Arroyo, I; Calzada-León, R; Morimoto, S; Téllez-Ascencio, N; Cerbón, M A

2002-03-01

The synthesis of dihydrotestosterone (DHT) is catalyzed by steroid 5alpha-reductase isozymes 1 and 2, and this function determines the development of the male phenotype during embriogenesis and the growth of androgen sensitive tissues during puberty. The aim of this study was to determine the cytosine methylation status of 5alpha-reductase isozymes types 1 and 2 genes in normal and in 5alpha-reductase deficient men. Genomic DNA was obtained from lymphocytes of both normal subjects and patients with primary 5alpha-reductase deficiency due to point mutations in 5alpha-reductase 2 gene. Southern blot analysis of 5alpha-reductase types 1 and 2 genes from DNA samples digested with HpaII presented a different cytosine methylation pattern compared to that observed with its isoschizomer MspI, indicating that both genes are methylated in CCGG sequences. The analysis of 5alpha-reductase 1 gene from DNA samples digested with Sau3AI and its isoschizomer MboI which recognize methylation in GATC sequences showed an identical methylation pattern. In contrast, 5alpha-reductase 2 gene digested with Sau3AI presented a different methylation pattern to that of the samples digested with MboI, indicating that steroid 5alpha-reductase 2 gene possess methylated cytosines in GATC sequences. Analysis of exon 4 of 5alpha-reductase 2 gene after metabisulfite PCR showed that normal and deficient subjects present a different methylation pattern, being more methylated in patients with 5alpha-reductase 2 mutated gene. The overall results suggest that 5alpha-reductase genes 1 and 2 are differentially methylated in lymphocytes from normal and 5alpha-reductase deficient patients. Moreover, the extensive cytosine methylation pattern observed in exon 4 of 5alpha-reductase 2 gene in deficient patients, points out to an increased rate of mutations in this gene.

The chorionic gonadotropin alpha-subunit gene is on human chromosome 18 in JEG cells.

PubMed Central

Hardin, J W; Riser, M E; Trent, J M; Kohler, P O

1983-01-01

The gene for the alpha subunit of human chorionic gonadotropin (hCG) has been tentatively assigned to human chromosome 18. This localization was accomplished through the use of Southern blot analysis. A full-length cDNA probe for the hCG alpha subunit and DNA isolated from a series of somatic hybrids between mouse and human cells were utilized to make this assignment. In addition, in situ hybridization with normal human peripheral blood lymphocytes as a source of human chromosomes and with the same cDNA probe confirmed this result. The presence of human chromosome 18 was required for the detection of DNA fragments characteristic of the alpha-hCG gene. These results are consistent with our previous observation that human chromosomes 10 and 18 are required for the production of hCG in cultured cells. Images PMID:6578509

Single-molecule analysis of DNA cross-links using nanopore technology

NASA Astrophysics Data System (ADS)

Wolna, Anna H.

The alpha-hemolysin (alpha-HL) protein ion channel is a potential next-generation sequencing platform that has been extensively used to study nucleic acids at a single-molecule level. After applying a potential across a lipid bilayer, the imbedded alpha-HL allows monitoring of the duration and current levels of DNA translocation and immobilization. Because this method does not require DNA amplification prior to sequencing, all the DNA damage present in the cell at any given time will be present during the sequencing experiment. The goal of this research is to determine if these damage sites give distinguishable current levels beyond those observed for the canonical nucleobases. Because DNA cross-links are one of the most prevalent types of DNA damage occurring in vivo, the blockage current levels were determined for thymine-dimers, guanine(C8)-thymine(N3) cross-links and platinum adducts. All of these cross-links give a different blockage current level compared to the undamaged strands when immobilized in the ion channel, and they all can easily translocate across the alpha-HL channel. Additionally, the alpha-HL nanopore technique presents a unique opportunity to study the effects of DNA cross-links, such as thymine-dimers, on the secondary structure of DNA G-quadruplexes folded from the human telomere sequence. Using this single-molecule nanopore technique we can detect subtle structural differences that cannot be easily addressed using conventional methods. The human telomere plays crucial roles in maintaining genome stability. In the presence of suitable cations, the repetitive 5'-TTAGGG human telomere sequence can fold into G-quadruplexes that adopt the hybrid fold in vivo. The telomere sequence is hypersensitive to UV-induced thymine-dimer (T=T) formation, and yet the presence of thymine dimers does not cause telomere shortening. The potential structural disruption and thermodynamic stability of the T=T-containing natural telomere sequences were studied to

Variability of Lyman-alpha emission from Jupiter

NASA Technical Reports Server (NTRS)

1979-01-01

The Jovian Lyman-alpha emission line was again observed in 1978 using the high resolution spectrometer on the Copernicus satellite. In intensity of 8.4+3.0 kilo Rayleighs was measured. This value represents a significant increase in intensity over previous (1976) Copernicus observations, but is lower than the recent (1979) values obtained by Voyager I and IUE. The increase in intensity was accompanied by a significant increase in line width, giving strong support to the theory that the emission results from resonant scattering of the solar Ly-alpha line by H atoms in the upper Jovian atmosphere. The strength of Jovian Ly-alpha emission correlates well with the level of solar activity. The solar extreme ultraviolet radiation varies with the solar cycle. This radiation causes the dissociation of H2 and CH4 into H atoms in the Jovian atmosphere. Therefore, in times of high solar activity, the H column density will increase, causing the observed stronger Jovian Ly-alpha emission.

Molecular interactions between single-stranded DNA-binding proteins associated with an essential MCAT element in the mouse smooth muscle alpha-actin promoter.

PubMed

Kelm, R J; Cogan, J G; Elder, P K; Strauch, A R; Getz, M J

1999-05-14

Transcriptional activity of the mouse vascular smooth muscle alpha-actin gene in fibroblasts is regulated, in part, by a 30-base pair asymmetric polypurine-polypyrimidine tract containing an essential MCAT enhancer motif. The double-stranded form of this sequence serves as a binding site for a transcription enhancer factor 1-related protein while the separated single strands interact with two distinct DNA binding activities termed VACssBF1 and 2 (Cogan, J. G., Sun, S., Stoflet, E. S., Schmidt, L. J., Getz, M. J., and Strauch, A. R. (1995) J. Biol. Chem. 270, 11310-11321; Sun, S., Stoflet, E. S., Cogan, J. G., Strauch, A. R., and Getz, M. J. (1995) Mol. Cell. Biol. 15, 2429-2936). VACssBF2 has been recently cloned and shown to consist of two closely related proteins, Puralpha and Purbeta (Kelm, R. J., Elder, P. K., Strauch, A. R., and Getz, M. J. (1997) J. Biol. Chem. 272, 26727-26733). In this study, we demonstrate that Puralpha and Purbeta interact with each other via highly specific protein-protein interactions and bind to the purine-rich strand of the MCAT enhancer in the form of both homo- and heteromeric complexes. Moreover, both Pur proteins interact with MSY1, a VACssBF1-like protein cloned by virtue of its affinity for the pyrimidine-rich strand of the enhancer. Interactions between Puralpha, Purbeta, and MSY1 do not require the participation of DNA. Combinatorial interactions between these three single-stranded DNA-binding proteins may be important in regulating activity of the smooth muscle alpha-actin MCAT enhancer in fibroblasts.

Methylation patterns of repetitive DNA sequences in germ cells of Mus musculus.

PubMed

Sanford, J; Forrester, L; Chapman, V; Chandley, A; Hastie, N

1984-03-26

The major and the minor satellite sequences of Mus musculus were undermethylated in both sperm and oocyte DNAs relative to the amount of undermethylation observed in adult somatic tissue DNA. This hypomethylation was specific for satellite sequences in sperm DNA. Dispersed repetitive and low copy sequences show a high degree of methylation in sperm DNA; however, a dispersed repetitive sequence was undermethylated in oocyte DNA. This finding suggests a difference in the amount of total genomic DNA methylation between sperm and oocyte DNA. The methylation levels of the minor satellite sequences did not change during spermiogenesis, and were not associated with the onset of meiosis or a specific stage in sperm development.

Formation of formaldehyde adducts in the reactions of DNA and deoxyribonucleosides with alpha-acetates of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), and N-nitrosodimethylamine (NDMA).

PubMed

Cheng, Guang; Wang, Mingyao; Upadhyaya, Pramod; Villalta, Peter W; Hecht, Stephen S

2008-03-01

The cytochrome P450-mediated alpha-hydroxylation of the carcinogenic nitrosamines N-nitrosodimethylamine (NDMA, 1), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, 6a), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL, 6b) produces diazonium ions and formaldehyde. The DNA-binding properties of the diazonium ions have been thoroughly characterized, and there is no doubt that they are critical in cancer induction by these nitrosamines. However, the possibility of additional DNA damage via released formaldehyde has not been reported. In this study, we used acetoxymethylmethylnitrosamine (5), 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (10a), and 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanol (10b) as stable precursors to the alpha-hydroxymethylnitrosamines that would be formed in the metabolism of NDMA, NNK, and NNAL. These alpha-acetates were incubated with calf thymus DNA in the presence of esterase at pH 7.0 and 37 degrees C. The DNA was isolated and enzymatically hydrolyzed to deoxyribonucleosides, and the hydrolysates were analyzed by liquid chromatography-electrospray ionization-mass spectrometry-selected ion monitoring for formaldehyde DNA adducts. Convincing evidence for the formation of the formaldehyde adducts N6-hydroxymethyl-dAdo (11), N4-hydroxymethyl-dCyd (12), N2-hydroxymethyl-dGuo (13), and the cross-links di-(N6-deoxyadenosyl)methane (14), (N6-deoxyadenosyl- N2-deoxyguanosyl)methane (15), and di-(N2-deoxyguanosyl)methane (16) was obtained in these reactions. These results demonstrate that NDMA, NNK, and NNAL have the potential to be bident carcinogens, damaging DNA through the metabolic formation of both diazonium ions and formaldehyde.

Molecular modeling of NK-CT1, from Indian monocellate cobra (Naja kaouthia) and its docking interaction with human DNA topoisomerase II alpha

PubMed Central

Bandopadhyay, Pathikrit; Halder, Soma; Sarkar, Mrinmoy; Kumar Bhunia, Sujay; Dey, Sananda; Gomes, Antony; Giri, Biplab

2016-01-01

A 6.76 kDa molecular weight cardio and cytotoxic protein of 60 amino acids in length called NK-CT1, was purified from the venom of Indian monocellate cobra (Naja kaouthia) by ion-exchange chromatography and HPLC as described in our earlier report. Therefore it is of interest to utlize the sequence of NK-CT1 for further functional inference using molecular modeling and docking. Thus homology model of NK-CT1 is described in this report. The anti-proliferative activity of the protein, binding with human DNA topoisomerase-II alpha was demonstrated using docking data with AUTODOCK and AUTODOCK MGL tools. Data shows that M26, V27 and S28 of NK-CT1 is in close contact with the nucleotides of the oligonucleotide, bound with topoisomerase-II alpha complex. PMID:28149043

The production and repair of aflatoxin B sub 1 -induced DNA damage

DOE Office of Scientific and Technical Information (OSTI.GOV)

Leadon, S.A.

To investigate the influence of function or activity of a DNA sequence on its repair, we have studied excision repair of aflatoxin B{sub 1} (AFB{sub 1})-induced damage in the nontranscribed, heterochromatic alpha DNA of monkey cells and in the metallothionein genes of human cells. In confluent cells, AFB{sub 1} adducts are produced in similar frequencies in alpha and in the rest of the DNA, but removal from alpha DNA is severely deficient, however, removal of AFB{sub 1} adducts from alpha DNA is enhanced by small doses of UV. The repair deficiencies are not observed in actively growing cells. We havemore » also shown that there is preferential repair of AFB{sub 1} damage in active genes. AFB{sub 1} damage is efficiently repaired in the active human metallothionein (hMT) genes, but deficiently repaired in inactive hMT genes. 51 refs., 3 tabs.« less

Transforming growth factor-alpha (TGF-alpha) in a semisolid dosage form: preservative and vehicle selection.

PubMed

Tan, E L; Shah, H S; Leister, K J; Kozick, L M; Pasciak, P; Vanderlaan, R K; Yu, C D; Patel, B

1993-08-01

The selection of an ideal semisolid vehicle for growth factors presents a challenge. Some antimicrobial agents are known to delay wound healing. The objective of this investigation was to identify appropriate preservatives and vehicles for TGF-alpha. Criteria for acceptance are noninterference with the mitogenic activity of TGF-alpha as well as adequate product preservation. Vehicles considered were o/w creams, ointments, and a gel. Combinations of six preservatives were tested. Selection was determined using both microbial preservative challenge and TGF-alpha mitogenic assay. In the former, 10 species of microorganisms were inoculated into formulation samples. At selected time intervals, it was determined whether colonies decreased, increased, or remained constant. In the mitogenic assay, samples of either preservatives or formulation prototypes were introduced to TGF-alpha-stimulated fibroblast cell cultures. Mitogenesis was determined by measuring 3H-dThd uptake into newly synthesized DNA. As preservatives, sorbic acid and quaternium-15 appear to satisfy both selection criteria. A thermosetting gel appears most promising as vehicle.

Cloning and purification of alpha-neurotoxins from king cobra (Ophiophagus hannah).

PubMed

He, Ying-Ying; Lee, Wei-Hui; Zhang, Yun

2004-09-01

Thirteen complete and three partial cDNA sequences were cloned from the constructed king cobra (Ophiophagus hannah) venom gland cDNA library. Phylogenetic analysis of nucleotide sequences of king cobra with those from other snake venoms revealed that obtained cDNAs are highly homologous to snake venom alpha-neurotoxins. Alignment of deduced mature peptide sequences of the obtained clones with those of other reported alpha-neurotoxins from the king cobra venom indicates that our obtained 16 clones belong to long-chain neurotoxins (seven), short-chain neurotoxins (seven), weak toxin (one) and variant (one), respectively. Up to now, two out of 16 newly cloned king cobra alpha-neurotoxins have identical amino acid sequences with CM-11 and Oh-6A/6B, which have been characterized from the same venom. Furthermore, five long-chain alpha-neurotoxins and two short-chain alpha-neurotoxins were purified from crude venom and their N-terminal amino acid sequences were determined. The cDNAs encoding the putative precursors of the purified native peptide were also determined based on the N-terminal amino acid sequencing. The purified alpha-neurotoxins showed different lethal activities on mice.

Far-Infrared and Millimeter Continuum Studies of K-Giants: Alpha Boo and Alpha Tau

NASA Technical Reports Server (NTRS)

Cohen, Martin; Carbon, Duane F.; Welch, William J.; Lim, Tanya; Forster, James R.; Goorvitch, David; Thigpen, William (Technical Monitor)

2002-01-01

We have imaged two normal, non-coronal, infrared-bright K-giants, alpha Boo and alpha Tau, in the 1.4-millimeter and 2.8-millimeter continuum using BIMA. These stars have been used as important absolute calibrators for several infrared satellites. Our goals are: (1) to probe the structure of their upper photospheres; (2) to establish whether these stars radiate as simple photospheres or possess long-wavelength chromospheres; and (3) to make a connection between millimeter-wave and far-infrared absolute flux calibrations. To accomplish these goals we also present ISO Long Wavelength Spectrometer (LWS) measurements of both these K-giants. The far-infrared and millimeter continuum radiation is produced in the vicinity of the temperature minimum in a Boo and a Tau, offering a direct test of the model photospheres and chromospheres for these two cool giants. We find that current photospheric models predict fluxes in reasonable agreement with those observed for those wavelengths which sample the upper photosphere, namely less than or equal to 170 micrometers in alpha Tau and less than or equal to 125 micrometers in alpha Boo. It is possible that alpha Tau is still radiative as far as 0.9 - 1.4 millimeters. We detect chromospheric radiation from both stars by 2.8 millimeters (by 1.4 millimeters in alpha Boo), and are able to establish useful bounds on the location of the temperature minimum. An attempt to interpret the chromospheric fluxes using the two-component "bifurcation model" proposed by Wiedemann et al. (1994) appears to lead to a significant contradiction.

Characterization of class II alpha genes and DLA-D region allelic associations in the dog.

PubMed

Sarmiento, U M; Storb, R F

1988-10-01

Human major histocompatibility complex (HLA) cDNA probes were used to analyze the restriction fragment length polymorphism (RFLP) of the alpha genes of the DLA-D region in dogs. Genomic DNA from peripheral blood leucocytes of 23 unrelated DLA-D homozygous dogs representing nine DLA-D types (defined by mixed leucocyte reaction) was digested with restriction enzymes (BamHI, EcoRI, Hind III, Pvu II, Taq I, Rsa I, Msp I, Pst I and Bgl II), separated by agarose gel electrophoresis and transferred onto Biotrace membrane. The Southern blots were successively hybridized with radiolabelled HLA cDNA probes corresponding to DQ, DP, DZ and DR alpha genes. Clear evidence was obtained for the canine homologues of DQ and DR alpha genes with simple bi- or tri-allelic polymorphism respectively. Evidence for a single, nonpolymorphic DP alpha gene was also obtained. However, the presence of a DZ alpha gene could not be clearly demonstrated in canine genomic DNA. This report extends our previous RFLP analysis documenting polymorphism of DLA class II beta genes in the same panel of homozygous typing cell dogs, and provides the basis for DLA-D genotyping at a population level. This study also characterizes the RFLP-defined preferential allelic associations across the DLA-D region in nine different homozygous typing cell specificities.

DNA polymerases in the rat pituitary gland. Effect of oestrogens and sulpiride.

PubMed

Jahn, G A; Kalbermann, L E; Machiavelli, G; Szijan, I; Burdman, J A

1980-06-01

Changes in the activity of DNA polymerase and [3H]thymidine incorporation into the DNA of the anterior pituitary gland were studied in oestrogenized male and pregnant rats. The activities of DNA polymerases alpha and beta, extracted in Tris--HCl or in sodium phosphate buffer were characterized according to their optimum pH and sensitivity to N-ethyl-maleimide. In the Tris-soluble fraction DNA polymerase activity is almost exclusively alpha, while in the phosphate soluble fraction it is a mixture of alpha and beta. The administration of oestrogens to male rats increases [3H]thymidine incorporation and enhances the activity of DNA polymerases in the Tris-soluble fraction, while the activity of the phosphate-soluble enzyme does not change. Sulpiride administration results in a further increment of [3H]thymidine incorporation and of DNA polymerase activity in the Tris-soluble fraction. In pregnant rats sulpiride also produces an increment of DNA polymerase activity only in the Tris-soluble fraction. Thus, the activity of the Tris-soluble fraction from APG behaves as DNA polymerase alpha. This activity changes in parallel with [3H]thymidine incorporation into DNA which is an indication of cell proliferation in the gland. This is discussed with respect to a negative feedback mechanism between intracellular prolactin concentration and DNA synthesis in the APG.

Development of a transmission alpha particle dosimetry technique using A549 cells and a Ra-223 source for targeted alpha therapy.

PubMed

Al Darwish, R; Staudacher, A H; Li, Y; Brown, M P; Bezak, E

2016-11-01

In targeted radionuclide therapy, regional tumors are targeted with radionuclides delivering therapeutic radiation doses. Targeted alpha therapy (TAT) is of particular interest due to its ability to deliver alpha particles of high linear energy transfer within the confines of the tumor. However, there is a lack of data related to alpha particle distribution in TAT. These data are required to more accurately estimate the absorbed dose on a cellular level. As a result, there is a need for a dosimeter that can estimate, or better yet determine the absorbed dose deposited by alpha particles in cells. In this study, as an initial step, the authors present a transmission dosimetry design for alpha particles using A549 lung carcinoma cells, an external alpha particle emitting source (radium 223; Ra-223) and a Timepix pixelated semiconductor detector. The dose delivery to the A549 lung carcinoma cell line from a Ra-223 source, considered to be an attractive radionuclide for alpha therapy, was investigated in the current work. A549 cells were either unirradiated (control) or irradiated for 12, 1, 2, or 3 h with alpha particles emitted from a Ra-223 source positioned below a monolayer of A549 cells. The Timepix detector was used to determine the number of transmitted alpha particles passing through the A549 cells and DNA double strand breaks (DSBs) in the form of γ-H2AX foci were examined by fluorescence microscopy. The number of transmitted alpha particles was correlated with the observed DNA DSBs and the delivered radiation dose was estimated. Additionally, the dose deposited was calculated using Monte Carlo code SRIM. Approximately 20% of alpha particles were transmitted and detected by Timepix. The frequency and number of γ-H2AX foci increased significantly following alpha particle irradiation as compared to unirradiated controls. The equivalent dose delivered to A549 cells was estimated to be approximately 0.66, 1.32, 2.53, and 3.96 Gy after 12, 1, 2, and 3 h

New insights into Trypanosoma cruzi evolution, genotyping and molecular diagnostics from satellite DNA sequence analysis.

PubMed

Ramírez, Juan C; Torres, Carolina; Curto, María de Los A; Schijman, Alejandro G

2017-12-01

Trypanosoma cruzi has been subdivided into seven Discrete Typing Units (DTUs), TcI-TcVI and Tcbat. Two major evolutionary models have been proposed to explain the origin of hybrid lineages, but while it is widely accepted that TcV and TcVI are the result of genetic exchange between TcII and TcIII strains, the origin of TcIII and TcIV is still a matter of debate. T. cruzi satellite DNA (SatDNA), comprised of 195 bp units organized in tandem repeats, from both TcV and TcVI stocks were found to have SatDNA copies type TcI and TcII; whereas contradictory results were observed for TcIII stocks and no TcIV sequence has been analyzed yet. Herein, we have gone deeper into this matter analyzing 335 distinct SatDNA sequences from 19 T. cruzi stocks representative of DTUs TcI-TcVI for phylogenetic inference. Bayesian phylogenetic tree showed that all sequences were grouped in three major clusters, which corresponded to sequences from DTUs TcI/III, TcII and TcIV; whereas TcV and TcVI stocks had two sets of sequences distributed into TcI/III and TcII clusters. As expected, the lowest genetic distances were found between TcI and TcIII, and between TcV and TcVI sequences; whereas the highest ones were observed between TcII and TcI/III, and among TcIV sequences and those from the remaining DTUs. In addition, signature patterns associated to specific T. cruzi lineages were identified and new primers that improved SatDNA-based qPCR sensitivity were designed. Our findings support the theory that TcIII is not the result of a hybridization event between TcI and TcII, and that TcIV had an independent origin from the other DTUs, contributing to clarifying the evolutionary history of T. cruzi lineages. Moreover, this work opens the possibility of typing samples from Chagas disease patients with low parasitic loads and improving molecular diagnostic methods of T. cruzi infection based on SatDNA sequence amplification.

Schindler disease: the molecular lesion in the alpha-N-acetylgalactosaminidase gene that causes an infantile neuroaxonal dystrophy.

PubMed Central

Wang, A M; Schindler, D; Desnick, R

1990-01-01

Schindler disease is a recently recognized infantile neuroaxonal dystrophy resulting from the deficient activity of the lysosomal hydrolase, alpha-N-acetylgalctosaminidase (alpha-GalNAc). The recent isolation and expression of the full-length cDNA encoding alpha-GalNAc facilitated the identification of the molecular lesions in the affected brothers from family D, the first cases described with this autosomal recessive disease. Southern and Northern hybridization analyses of DNA and RNA from the affected homozygotes revealed a grossly normal alpha-GalNAc gene structure and normal transcript sizes and amounts. Therefore, the alpha-GalNAc transcript from an affected homozygote was reverse-transcribed, amplified by the polymerase chain reaction (PCR), and sequenced. A single G to A transition at nucleotide 973 was detected in multiple subclones containing the PCR products. This point mutation resulted in a glutamic acid to lysine substitution in residue 325 (E325K) of the alpha-GalNAc polypeptide. The base substitution was confirmed by dot blot hybridization analyses of PCR-amplified genomic DNA from family members with allele-specific oligonucleotides. Furthermore, transient expression of an alpha-GalNAc construct containing the E325K mutation resulted in the expression of an immunoreactive polypeptide which had no detectable alpha-GalNAc activity. Images PMID:2243144

Lyman-alpha observations of Comet Kohoutek 1973 XII with Copernicus

NASA Technical Reports Server (NTRS)

Drake, J. F.; Jenkins, E. B.; Bertaux, J. L.; Festou, M.; Keller, H. U.

1976-01-01

Comet Kohoutek 1973 XII was observed with a telescope-spectrometer on the Copernicus satellite on six occasions over a 1-month period starting on January 29, 1974. Positive detection of the cometary Ly-alpha emission profile was obtained on January 29 and February 2. Earlier observations of the geocoronal Ly-alpha emission profile allowed an instrumental intensity calibration and confirmation of the computed instrumental profile for an extended source at the Ly-alpha wavelength. After allowing for broadening by the instrument, a hydrogen-outflow velocity of about 10.6 km/s is derived from the width of the Ly-alpha emission on January 29. The intensity calibration combined with an appropriate cometary model led to cometary water-production rates for January 29 and February 2. Only upper limits were obtained for Ly-alpha on and after February 14. Searches for OH and D led to negative results.

A Pilot Study of Noninvasive Prenatal Diagnosis of Alpha- and Beta-Thalassemia with Target Capture Sequencing of Cell-Free Fetal DNA in Maternal Blood.

PubMed

Wang, Wenjuan; Yuan, Yuan; Zheng, Haiqing; Wang, Yaoshen; Zeng, Dan; Yang, Yihua; Yi, Xin; Xia, Yang; Zhu, Chunjiang

2017-07-01

Thalassemia is a dangerous hematolytic genetic disease. In south China, ∼24% Chinese carry alpha-thalassemia or beta-thalassemia gene mutations. Given the fact that the invasive sampling procedures can only be performed by professionals in experienced centers, it may increase the risk of miscarriage or infection. Thus, most people are worried about the invasive operation. As such, a noninvasive and accurate prenatal diagnosis is needed for appropriate genetic counseling for families with high risks. Here we sought to develop capture probes and their companion analysis methods for the noninvasive prenatal detection of deletional and nondeletional thalassemia. Two families diagnosed as carriers of either beta-thalassemia gene or Southeast Asian deletional alpha-thalassemia gene mutation were recruited. The maternal plasma and amniotic fluid were collected for prenatal diagnosis. Probes targeting exons of the genes of interest and the highly heterozygous SNPs within the 1Mb flanking region were designed. The target capture sequencing was performed with plasma DNA from the pregnant woman and genomic DNA from the couples and their children. Then the parental haplotype was constructed by the trios-based strategy. The fetal haplotype was deduced from the parental haplotype with a hidden Markov model-based algorithm. The fetal genotypes were successfully deduced in both families noninvasively. The noninvasively constructed haplotypes of both fetuses were identical to the invasive prenatal diagnosis results with an accuracy rate of 100% in the target region. Our study demonstrates that the effective noninvasive prenatal diagnosis of alpha-thalassemia and beta-thalassemia can be achieved with the targeted capture sequencing and the haplotype-assisted analysis method.

Major satellite repeat RNA stabilize heterochromatin retention of Suv39h enzymes by RNA-nucleosome association and RNA:DNA hybrid formation.

PubMed

Velazquez Camacho, Oscar; Galan, Carmen; Swist-Rosowska, Kalina; Ching, Reagan; Gamalinda, Michael; Karabiber, Fethullah; De La Rosa-Velazquez, Inti; Engist, Bettina; Koschorz, Birgit; Shukeir, Nicholas; Onishi-Seebacher, Megumi; van de Nobelen, Suzanne; Jenuwein, Thomas

2017-08-01

The Suv39h1 and Suv39h2 histone lysine methyltransferases are hallmark enzymes at mammalian heterochromatin. We show here that the mouse Suv39h2 enzyme differs from Suv39h1 by containing an N-terminal basic domain that facilitates retention at mitotic chromatin and provides an additional affinity for major satellite repeat RNA. To analyze an RNA-dependent interaction with chromatin, we purified native nucleosomes from mouse ES cells and detect that Suv39h1 and Suv39h2 exclusively associate with poly-nucleosomes. This association was attenuated upon RNaseH incubation and entirely lost upon RNaseA digestion of native chromatin. Major satellite repeat transcripts remain chromatin-associated and have a secondary structure that favors RNA:DNA hybrid formation. Together, these data reveal an RNA-mediated mechanism for the stable chromatin interaction of the Suv39h KMT and suggest a function for major satellite non-coding RNA in the organization of an RNA-nucleosome scaffold as the underlying structure of mouse heterochromatin.

Variability of Lyman-alpha emission from Jupiter

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cochran, W.D.; Barker, E.S.

1979-12-01

The Jovian L..cap alpha.. emission line was reobserved in 1978 March using the high-resolution spectrometer of the Copernicus satellite. An intensity of 8.3 +- 2.9 kilorayleighs was measured. This value represents a significant increase in intensity over previous (1976) Copernicus observations, but is lower than the recent (1979) values obtained by Voyager 1 and IUE. The increase in intensity has been accompanied by a significant increase in line width givin strong support to the theory that the emission results from resonant scattering of the solar L..cap alpha.. line by H atoms in the upper Jovian atmosphere. The strength of Jovianmore » L..cap alpha.. emission correlates well with the level of solar activity. The solar extreme ultraviolet radiation varies with the solar cycle. This radiation causes the dissociation of H/sub 2/ and CH/sub 4/ into H atoms in the Jovian atmosphere. Therefore, in times of high solar activity, the H column density will increase, causing the observed stronger Jovian L..cap alpha.. emission.« less

Transcription of tandemly repetitive DNA: functional roles.

PubMed

Biscotti, Maria Assunta; Canapa, Adriana; Forconi, Mariko; Olmo, Ettore; Barucca, Marco

2015-09-01

A considerable fraction of the eukaryotic genome is made up of satellite DNA constituted of tandemly repeated sequences. These elements are mainly located at centromeres, pericentromeres, and telomeres and are major components of constitutive heterochromatin. Although originally satellite DNA was thought silent and inert, an increasing number of studies are providing evidence on its transcriptional activity supporting, on the contrary, an unexpected dynamicity. This review summarizes the multiple structural roles of satellite noncoding RNAs at chromosome level. Indeed, satellite noncoding RNAs play a role in the establishment of a heterochromatic state at centromere and telomere. These highly condensed structures are indispensable to preserve chromosome integrity and genome stability, preventing recombination events, and ensuring the correct chromosome pairing and segregation. Moreover, these RNA molecules seem to be involved also in maintaining centromere identity and in elongation, capping, and replication of telomere. Finally, the abnormal variation of centromeric and pericentromeric DNA transcription across major eukaryotic lineages in stress condition and disease has evidenced the critical role that these transcripts may play and the potentially dire consequences for the organism.

The yeast Saccharomyces cerevisiae DNA polymerase IV: possible involvement in double strand break DNA repair.

PubMed

Leem, S H; Ropp, P A; Sugino, A

1994-08-11

We identified and purified a new DNA polymerase (DNA polymerase IV), which is similar to mammalian DNA polymerase beta, from Saccharomyces cerevisiae and suggested that it is encoded by YCR14C (POLX) on chromosome III. Here, we provided a direct evidence that the purified DNA polymerase IV is indeed encoded by POLX. Strains harboring a pol4 deletion mutation exhibit neither mitotic growth defect nor a meiosis defect, suggesting that DNA polymerase IV participates in nonessential functions in DNA metabolism. The deletion strains did not exhibit UV-sensitivity. However, they did show weak sensitivity to MMS-treatment and exhibited a hyper-recombination phenotype when intragenic recombination was measured during meiosis. Furthermore, MAT alpha pol4 delta segregants had a higher frequency of illegitimate mating with a MAT alpha tester strain than that of wild-type cells. These results suggest that DNA polymerase IV participates in a double-strand break repair pathway. A 3.2kb of the POL4 transcript was weakly expressed in mitotically growing cells. During meiosis, a 2.2 kb POL4 transcript was greatly induced, while the 3.2 kb transcript stayed at constant levels. This induction was delayed in a swi4 delta strain during meiosis, while no effect was observed in a swi6 delta strain.

[Cloning of Chinese Banna minipig inbred-line alpha1,3-galactosyltransferase gene and construction of its recombinant eukaryotic expression vector].

PubMed

Zhu, Shengming; Wang, Yanping; Zheng, Hong; Cheng, Jingqiu; Lu, Yanrong; Zeng, Yangzhi; Wang, Yu; Wang, Zhu

2009-04-01

This study sought to clone Chinese Banna minipig inbred-line (BMI) alpha1,3-galactosyltransferase (alpha1,3-GT) gene and construct its recombinant eukaryotic expression vector. Total RNA was isolated from BMI liver. Full length cDNA of alpha1,3-GT gene was amplified by RT-PCR and cloned into pMD18-T vector to sequence. Subsequently, alpha1,3-GT gene was inserted into pEGFP-N1 to construct eukaryotic expression vector pEGFP-N1-GT. Then the reconstructed plasmid pEGFP-N1-GT was transiently transfected into human lung cancer cell line A549. The expression of alpha1,3-GT mRNA in transfected cells was detected by RT-PCR. FITC-BS-IB4 lectin was used in the direct immunofluorescence method, which was performed to observe the alpha-Gal synthesis function of BMI alpha1,3-GT in transfected cells. The results showed that full length of BMI alpha1,3-GT cDNA was 1116 bp. BMI alpha1,3-GT cDNA sequence was highly homogenous with those of mouse and bovine, and was exactly the same as the complete sequence of those of swine, pEGFP-N1-GT was confirmed by enzyme digestion and PCR. The expression of alpha1,3-GT mRNA was detected in A549 cells transfected by pEGFP-N1-GT. The expression of alpha-Gal was observed on the membrane of A549 cells transfected by pEGFP-N1-GT. Successful cloning of BMI alpha1,3-GT cDNA and construction of its eukaryotic expression vector have established a foundation for further research and application of BMI alpha1,3-GT in the fields of xenotransplantation and immunological therapy of cancer.

A novel subviral agent associated with a geminivirus: The first report of a DNA satellite

PubMed Central

Dry, Ian B.; Krake, Leslie R.; Rigden, Justin E.; Rezaian, M. Ali

1997-01-01

Numerous plant RNA viruses have associated with them satellite (sat) RNAs that have little or no nucleotide sequence similarity to either the viral or host genomes but are completely dependent on the helper virus for replication. We report here on the discovery of a 682-nt circular DNA satellite associated with tomato leaf curl geminivirus (TLCV) infection in northern Australia. This is the first demonstration that satellite molecules are not limited to RNA viral systems. The DNA satellite (TLCV sat-DNA) is strictly dependent for replication on the helper virus replication-associated protein and is encapsidated by TLCV coat protein. It has no significant open reading frames, and it shows no significant sequence similarity to the 2766-nt helper-virus genome except for two short motifs present in separate putative stem–loop structures: TAATATTAC, which is universally conserved in all geminiviruses, and AATCGGTGTC, which is identical to a putative replication-associated protein binding motif in TLCV. Replication of TLCV sat-DNA is also supported by other taxonomically distinct geminiviruses, including tomato yellow leaf curl virus, African cassava mosaic virus, and beet curly top virus. Therefore, this unique DNA satellite does not appear to strictly conform with the requirements that dictate the specificity of interaction of geminiviral replication-associated proteins with their cognate origins as predicted by the current model of geminivirus replication. PMID:9192696

DNA Repair, Redox Regulation and Modulation of Estrogen Receptor Alpha Mediated Transcription

ERIC Educational Resources Information Center

Curtis-Ducey, Carol Dianne

2009-01-01

Interaction of estrogen receptor [alpha] (ER[alpha]) with 17[beta]-estradiol (E[subscript 2]) facilitates binding of the receptor to estrogen response elements (EREs) in target genes, which in turn leads to recruitment of coregulatory proteins. To better understand how estrogen-responsive genes are regulated, our laboratory identified a number of…

First evidence of DNA methylation in insect Tribolium castaneum: environmental regulation of DNA methylation within heterochromatin.

PubMed

Feliciello, Isidoro; Parazajder, Josip; Akrap, Ivana; Ugarković, Durđica

2013-05-01

DNA methylation has been studied in many eukaryotic organisms, in particular vertebrates, and was implicated in developmental and phenotypic variations. Little is known about the role of DNA methylation in invertebrates, although insects are considered as excellent models for studying the evolution of DNA methylation. In the red flour beetle, Tribolium castaneum (Tenebrionidae, Coleoptera), no evidence of DNA methylation has been found till now. In this paper, a cytosine methylation in Tribolium castaneum embryos was detected by methylation sensitive restriction endonucleases and immuno-dot blot assay. DNA methylation in embryos is followed by a global demethylation in larvae, pupae and adults. DNA demethylation seems to proceed actively through 5-hydroxymethylcytosine, most probably by the action of TET enzyme. Bisulfite sequencing of a highly abundant satellite DNA located in pericentromeric heterochromatin revealed similar profile of cytosine methylation in adults and embryos. Cytosine methylation was not only restricted to CpG sites but was found at CpA, CpT and CpC sites. In addition, complete cytosine demethylation of heterochromatic satellite DNA was induced by heat stress. The results reveal existence of DNA methylation cycling in T. castaneum ranging from strong overall cytosine methylation in embryos to a weak DNA methylation in other developmental stages. Nevertheless, DNA methylation is preserved within heterochromatin during development, indicating its role in heterochromatin formation and maintenance. It is, however, strongly affected by heat stress, suggesting a role for DNA methylation in heterochromatin structure modulation during heat stress response.

Creatine kinase and alpha-actin mRNA levels decrease in diabetic rat hearts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Popovich, B.; Barrieux, A.; Dillmann, W.H.

1987-05-01

Diabetic cardiomyopathy is associated with cardiac atrophy and isoenzyme redistribution. To determine if tissue specific changes occur in mRNAs coding for ..cap alpha..-actin and creatine kinase (CK), they performed RNA blot analysis. Total ventricular RNA from control (C) and 4 wk old diabetic (D) rats were hybridized with /sup 32/P cDNA probes for ..cap alpha..-actin and CK. A tissue independent cDNA probe, CHOA was also used. Signal intensity was quantified by photodensitometry. D CK mRNA was 47 +/- 16% lower in D vs C. Insulin increases CK mRNA by 20% at 1.5 hs, and completely reverses the deficit after 4more » wks. D ..cap alpha..-actin mRNA is 66 +/- 18% lower in D vs C. Insulin normalized ..cap alpha..-actin mRNA by 5 hs. CHOA mRNA is unchanged in D vs C, but D + insulin CHOA mRNA is 30 +/- 2% lower than C. In rats with diabetic cardiomyopathy, muscle specific CK and ..cap alpha..-actin mRNAs are decreased. Insulin treatment reverses these changes.« less

Major satellite repeat RNA stabilize heterochromatin retention of Suv39h enzymes by RNA-nucleosome association and RNA:DNA hybrid formation

PubMed Central

Velazquez Camacho, Oscar; Galan, Carmen; Swist-Rosowska, Kalina; Ching, Reagan; Gamalinda, Michael; Karabiber, Fethullah; De La Rosa-Velazquez, Inti; Engist, Bettina; Koschorz, Birgit; Shukeir, Nicholas; Onishi-Seebacher, Megumi; van de Nobelen, Suzanne; Jenuwein, Thomas

2017-01-01

The Suv39h1 and Suv39h2 histone lysine methyltransferases are hallmark enzymes at mammalian heterochromatin. We show here that the mouse Suv39h2 enzyme differs from Suv39h1 by containing an N-terminal basic domain that facilitates retention at mitotic chromatin and provides an additional affinity for major satellite repeat RNA. To analyze an RNA-dependent interaction with chromatin, we purified native nucleosomes from mouse ES cells and detect that Suv39h1 and Suv39h2 exclusively associate with poly-nucleosomes. This association was attenuated upon RNaseH incubation and entirely lost upon RNaseA digestion of native chromatin. Major satellite repeat transcripts remain chromatin-associated and have a secondary structure that favors RNA:DNA hybrid formation. Together, these data reveal an RNA-mediated mechanism for the stable chromatin interaction of the Suv39h KMT and suggest a function for major satellite non-coding RNA in the organization of an RNA-nucleosome scaffold as the underlying structure of mouse heterochromatin. DOI: http://dx.doi.org/10.7554/eLife.25293.001 PMID:28760199

Dielectronic satellite spectra of hydrogen-like titanium (Ti XXII)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bitter, M.; von Goeler, S.; Cohen, S.

High resolution spectra of the Ly ..cap alpha../sub 1/ and Ly ..cap alpha../sub 2/ lines of hydrogenlike titanium, TiXXII, and the associated dielectronic satellites which are due to transitions 1snl-2pnl with n greater than or equal to 2, have been observed from tokamak discharges with auxiliary ion cyclotron heating (ICRH) with central electron temperatures of 2 keV and central electron densities of 8 x 10/sup 13/ cm/sup -3/ on the Princeton Large Torus (PLT). The data have been used for a detailed comparison with theoretical predictions based on the Z - expansion method and Hartree - Fock calculations. The resultsmore » obtained with the Z - expansion method are in excellent agreement with the observed spectral data except for minor discrepancies between the theoretical and experimental wavelengths of 0.0003 A for the n = 2 satellites and of 0.0001 A for the separation of the Ly ..cap alpha../sub 1/ and Ly ..cap alpha../sub 2/ lines. Very good agreement with the experimental data is also obtained for the results from the Hartree - Fock calculations though somewhat larger discrepancies (approx. = 0.0009 A) exist between experimental and theoretical wavelengths which are systematically too small. The observed spectra are used for diagnosis of the central ion and electron temperatures of the PLT discharges and for a measurement of the dielectronic recombination rate coefficient of TiXXII.« less

Implications of the dependence of the elastic properties of DNA on nucleotide sequence.

PubMed

Olson, Wilma K; Swigon, David; Coleman, Bernard D

2004-07-15

Recent advances in structural biochemistry have provided evidence that not only the geometric properties but also the elastic moduli of duplex DNA are strongly dependent on nucleotide sequence in a way that is not accounted for by classical rod models of the Kirchhoff type. A theory of sequence-dependent DNA elasticity is employed here to calculate the dependence of the equilibrium configurations of circular DNA on the binding of ligands that can induce changes in intrinsic twist at a single base-pair step. Calculations are presented of the influence on configurations of the assumed values and distribution along the DNA of intrinsic roll and twist and a modulus coupling roll to twist. Among the results obtained are the following. For minicircles formed from intrinsically straight DNA, the distribution of roll-twist coupling strongly affects the dependence of the total elastic energy Psi on the amount alpha of imposed untwisting, and that dependence can be far from quadratic. (In fact, for a periodic distribution of roll-twist coupling with a period equal to the intrinsic helical repeat length, Psi can be essentially independent of alpha for -90 degrees < alpha <90 degrees.) When the minicircle is homogeneous and without roll-twist coupling, but with uniform positive intrinsic roll, the point at which Psi attains its minimum value shifts towards negative values of alpha. It is remarked that there are cases in which one can relate graphs of Psi versus alpha to the 'effective values' of bending and twisting moduli and helical repeat length obtained from measurements of equilibrium distributions of topoisomers and probabilities of ring closure. For a minicircle formed from DNA that has an 'S' shape when stress-free, the graphs of Psi versus alpha have maxima at alpha = 0. As the binding of a twisting agent to such a minicircle results in a net decrease in Psi, the affinity of the twisting agent for binding to the minicircle is greater than its affinity for binding to

Transcription of highly repetitive tandemly organized DNA in amphibians and birds: A historical overview and modern concepts.

PubMed

Trofimova, Irina; Krasikova, Alla

2016-12-01

Tandemly organized highly repetitive DNA sequences are crucial structural and functional elements of eukaryotic genomes. Despite extensive evidence, satellite DNA remains an enigmatic part of the eukaryotic genome, with biological role and significance of tandem repeat transcripts remaining rather obscure. Data on tandem repeats transcription in amphibian and avian model organisms is fragmentary despite their genomes being thoroughly characterized. Review systematically covers historical and modern data on transcription of amphibian and avian satellite DNA in somatic cells and during meiosis when chromosomes acquire special lampbrush form. We highlight how transcription of tandemly repetitive DNA sequences is organized in interphase nucleus and on lampbrush chromosomes. We offer LTR-activation hypotheses of widespread satellite DNA transcription initiation during oogenesis. Recent explanations are provided for the significance of high-yield production of non-coding RNA derived from tandemly organized highly repetitive DNA. In many cases the data on the transcription of satellite DNA can be extrapolated from lampbrush chromosomes to interphase chromosomes. Lampbrush chromosomes with applied novel technical approaches such as superresolution imaging, chromosome microdissection followed by high-throughput sequencing, dynamic observation in life-like conditions provide amazing opportunities for investigation mechanisms of the satellite DNA transcription.

Transcription of highly repetitive tandemly organized DNA in amphibians and birds: A historical overview and modern concepts

PubMed Central

Krasikova, Alla

2016-01-01

ABSTRACT Tandemly organized highly repetitive DNA sequences are crucial structural and functional elements of eukaryotic genomes. Despite extensive evidence, satellite DNA remains an enigmatic part of the eukaryotic genome, with biological role and significance of tandem repeat transcripts remaining rather obscure. Data on tandem repeats transcription in amphibian and avian model organisms is fragmentary despite their genomes being thoroughly characterized. Review systematically covers historical and modern data on transcription of amphibian and avian satellite DNA in somatic cells and during meiosis when chromosomes acquire special lampbrush form. We highlight how transcription of tandemly repetitive DNA sequences is organized in interphase nucleus and on lampbrush chromosomes. We offer LTR-activation hypotheses of widespread satellite DNA transcription initiation during oogenesis. Recent explanations are provided for the significance of high-yield production of non-coding RNA derived from tandemly organized highly repetitive DNA. In many cases the data on the transcription of satellite DNA can be extrapolated from lampbrush chromosomes to interphase chromosomes. Lampbrush chromosomes with applied novel technical approaches such as superresolution imaging, chromosome microdissection followed by high-throughput sequencing, dynamic observation in life-like conditions provide amazing opportunities for investigation mechanisms of the satellite DNA transcription. PMID:27763817

Identification of a GTP-binding protein. cap alpha. subunit that lacks an apparent ADP-ribosylation site for pertussis toxin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fong, H.K.W.; Yoshimoto, K.K.; Eversole-Cire, P.

1988-05-01

Recent molecular cloning of cDNA for the ..cap alpha.. subunit of bovine transducin (a guanine nucleotide-binding regulatory protein, or G protein) has revealed the presence of two retinal-specific transducins, called T/sub r/ and T/sub c/, which are expressed in rod or cone photoreceptor cells. In a further study of G-protein diversity and signal transduction in the retina, the authors have identified a G-protein ..cap alpha.. subunit, which they refer to as G/sub z/..cap alpha.., by isolating a human retinal cDNA clone that cross-hybridizes at reduced stringency with bovine T/sub r/ ..cap alpha..-subunit cDNA. The deduced amino acid sequence of G/submore » z/..cap alpha.. is 41-67% identical with those of other known G-protein ..cap alpha.. subunits. However, the 355-residue G/sub z/..cap alpha.. lacks a consensus site for ADP-ribosylation by pertussis toxin, and its amino acid sequence varies within a number of regions that are strongly conserved among all of the other G-protein ..cap alpha.. subunits. They suggest that G/sub z/..cap alpha.., which appears to be highly expressed in neural tissues, represents a member of a subfamily of G proteins that mediate signal transduction in pertussis toxin-insensitive systems.« less

TNF-{alpha} promotes cell survival through stimulation of K{sup +} channel and NF{kappa}B activity in corneal epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang Ling; Reinach, Peter; Lu, Luo

2005-11-15

Tumor necrosis factor (TNF-{alpha}) in various cell types induces either cell death or mitogenesis through different signaling pathways. In the present study, we determined in human corneal epithelial cells how TNF-{alpha} also promotes cell survival. Human corneal epithelial (HCE) cells were cultured in DMEM/F-12 medium containing 10% FBS. TNF-{alpha} stimulation induced activation of a voltage-gated K{sup +} channel detected by measuring single channel activity using patch clamp techniques. The effect of TNF-{alpha} on downstream events included NF{kappa}B nuclear translocation and increases in DNA binding activities, but did not elicit ERK, JNK, or p38 limb signaling activation. TNF-{alpha} induced increases inmore » p21 expression resulting in partial cell cycle attenuation in the G{sub 1} phase. Cell cycle progression was also mapped by flow cytometer analysis. Blockade of TNF-{alpha}-induced K{sup +} channel activity effectively prevented NF{kappa}B nuclear translocation and binding to DNA, diminishing the cell-survival protective effect of TNF-{alpha}. In conclusion, TNF-{alpha} promotes survival of HCE cells through sequential stimulation of K{sup +} channel and NF{kappa}B activities. This response to TNF-{alpha} is dependent on stimulating K{sup +} channel activity because following suppression of K{sup +} channel activity TNF-{alpha} failed to activate NF{kappa}B nuclear translocation and binding to nuclear DNA.« less

Diversity of Dicotyledenous-Infecting Geminiviruses and Their Associated DNA Molecules in Southern Africa, Including the South-West Indian Ocean Islands

PubMed Central

Rey, Marie E. C.; Ndunguru, Joseph; Berrie, Leigh C.; Paximadis, Maria; Berry, Shaun; Cossa, Nurbibi; Nuaila, Valter N.; Mabasa, Ken G.; Abraham, Natasha; Rybicki, Edward P.; Martin, Darren; Pietersen, Gerhard; Esterhuizen, Lindy L.

2012-01-01

The family Geminiviridae comprises a group of plant-infecting circular ssDNA viruses that severely constrain agricultural production throughout the temperate regions of the world, and are a particularly serious threat to food security in sub-Saharan Africa. While geminiviruses exhibit considerable diversity in terms of their nucleotide sequences, genome structures, host ranges and insect vectors, the best characterised and economically most important of these viruses are those in the genus Begomovirus. Whereas begomoviruses are generally considered to be either monopartite (one ssDNA component) or bipartite (two circular ssDNA components called DNA-A and DNA-B), many apparently monopartite begomoviruses are associated with additional subviral ssDNA satellite components, called alpha- (DNA-αs) or betasatellites (DNA-βs). Additionally, subgenomic molecules, also known as defective interfering (DIs) DNAs that are usually derived from the parent helper virus through deletions of parts of its genome, are also associated with bipartite and monopartite begomoviruses. The past three decades have witnessed the emergence and diversification of various new begomoviral species and associated DI DNAs, in southern Africa, East Africa, and proximal Indian Ocean islands, which today threaten important vegetable and commercial crops such as, tobacco, cassava, tomato, sweet potato, and beans. This review aims to describe what is known about these viruses and their impacts on sustainable production in this sensitive region of the world. PMID:23170182

Extended atmospheres of outer planet satellites and comets

NASA Technical Reports Server (NTRS)

Smyth, W. H.; Combi, M. R.

1985-01-01

Model analysis of the extended atmospheres of outer planet satellites and comets are discussed. Understanding the neutral hydrogen distribution in the Saturn system concentrated on assessing the spatial dependence of the lifetime of hydrogen atoms and on obtaining appropriately sorted Lyman ALPHA data from the Voyager 1 UVS instrument. Progress in the area of the extended cometary atmospheres included analysis of Pioneer Venus Layman alpha observations of Comet P/Encke with the fully refined hydrogen cloud model, development of the basic carbon and oxygen models, and planning for the Pioneer Venus UVS observations of Comets P/Giacobini-Zinner and P/Halley.

Apoptosis induced by tumor necrosis factor-alpha in rat hepatocyte cell lines expressing hepatitis B virus.

PubMed Central

Guilhot, S.; Miller, T.; Cornman, G.; Isom, H. C.

1996-01-01

Three well differentiated SV40-immortalized rat hepatocyte cell lines, CWSV1, CWSV2, and CWSV14, and Hepatitis B Virus (HBV)-producing cell lines derived from them were examined for sensitivity to tumor necrosis factor (TNF)-alpha. CWSV1, CWSV2, and CWSV14 cells were co-transfected with a DNA construct containing a dimer of the HBV genome and the neo gene and selected in G418 to generate stable cell lines. Characterization of these cell lines indicated that they contain integrated HBV DNA, contain low molecular weight HBV DNA compatible with the presence of HBV replication intermediates, express HBV transcripts, and produce HBV proteins. The viability of CWSV1, CWSV2, and CWSV2 cells was not significantly altered when they were treated with TNF-alpha at concentrations as high as 20,000 U/ml. The HBV-expressing CWSV1 cell line, SV1di36, and the HBV-expressing CWSV14 cell line, SV14di208, were also not killed when treated with TNF-alpha. However, the HBV-expressing CWSV2 cell line, SV2di366, was extensively killed when treated with TNF-alpha at concentrations ranging from 200 to 20,000 U/ml. Analysis of several different HBV-producing CWSV2 cell lines indicated that TNF-alpha killing depended upon the level of HBV expression. The TNF-alpha-induced cell killing in high HBV-producing CWSV2 cell lines was accompanied by the presence of an oligonucleosomal DNA ladder characteristic of apoptosis. Images Figure 2 Figure 3 Figure 4 Figure 6 Figure 9 Figure 10 Figure 11 PMID:8774135

X-ray spectroscopy of SNR E0102-72 with the ASCA satellite

NASA Technical Reports Server (NTRS)

Hayashi, Ichizo; Koyama, Katsuji; Ozaki, Masanobu; Miyata, Emi; Tsumeni, Hiroshi; Hughes, John P.; Petre, Robert

1994-01-01

The Advanced Satellite for Cosmology and Astrophysics (ASCA) satellite has obtained a moderate-resolution energy spectrum of E0102-72, the brightest Supernova Remnant (SNR) in the Small Magellanic Cloud (SMC). This paper reports on the first results of the analysis of the high quality spectrum of E0102-72. The spectrum shows resolved emission lines of He-like K alpha, H-like K alpha and K beta from oxygen, neon, and magnesium. The intensity ratios of these lines cannot be explained by a multi-component plasma model with uniform abundances, but requires abundance inhomogeneity in the plasma. We demonstrate how the spectral capabilities of the ASCA SIS make available new diagnostics of X-ray plasmas in a state of non-equilibrium ionization. Some interpretation based on the spectral analysis is also given.

Single-molecule study of thymidine glycol and i-motif through the alpha-hemolysin ion channel

NASA Astrophysics Data System (ADS)

He, Lidong

Nanopore-based devices have emerged as a single-molecule detection and analysis tool for a wide range of applications. Through electrophoretically driving DNA molecules across a nanosized pore, a lot of information can be received, including unfolding kinetics and DNA-protein interactions. This single-molecule method has the potential to sequence kilobase length DNA polymers without amplification or labeling, approaching "the third generation" genome sequencing for around $1000 within 24 hours. alpha-Hemolysin biological nanopores have the advantages of excellent stability, low-noise level, and precise site-directed mutagenesis for engineering this protein nanopore. The first work presented in this thesis established the current signal of the thymidine glycol lesion in DNA oligomers through an immobilization experiment. The thymidine glycol enantiomers were differentiated from each other by different current blockage levels. Also, the effect of bulky hydrophobic adducts to the current blockage was investigated. Secondly, the alpha-hemolysin nanopore was used to study the human telomere i-motif and RET oncogene i-motif at a single-molecule level. In Chapter 3, it was demonstrated that the alpha-hemolysin nanopore can differentiate an i-motif form and single-strand DNA form at different pH values based on the same sequence. In addition, it shows potential to differentiate the folding topologies generated from the same DNA sequence.

Observations of interstellar hydrogen and deuterium toward Alpha Centauri A

NASA Technical Reports Server (NTRS)

Landsman, W. B.; Henry, R. C.; Moos, H. W.; Linsky, J. L.

1984-01-01

A composite profile is presented of the Ly-alpha emission line of Alpha Cen A, obtained from 10 individual spectra with the high-resolution spectrograph aboard the International Ultraviolet Explorer (IUE) satellite. There is excellent overall agreement with two previous Copernicus observations. Interstellar deuterium is detected, and a lower limit is set on the deuterium to hydrogen ratio of nDI/nHI greater than 8 x 10 to the -6th. In addition, the deuterium bulk velocity appears blueshifted by 8 + or - 2 km/s with respect to interstellar hydrogen, suggesting a nonuniform medium along the line of sight.

Human alpha 7 acetylcholine receptor: cloning of the alpha 7 subunit from the SH-SY5Y cell line and determination of pharmacological properties of native receptors and functional alpha 7 homomers expressed in Xenopus oocytes.

PubMed

Peng, X; Katz, M; Gerzanich, V; Anand, R; Lindstrom, J

1994-03-01

The alpha-bungarotoxin-binding acetylcholine receptors from the human neuroblastoma cell line SH-SY5Y were found to cross-react with some monoclonal antibodies to alpha 7 subunits of nicotinic acetylcholine receptors from chicken brain. The human alpha 7 subunit cDNA from SH-SY5Y was cloned, revealing 94% amino acid sequence identity to rat alpha 7 subunits and 92% identity to chicken alpha 7 subunits. Native human alpha 7 receptors showed affinities for some ligands similar to those previously observed with native chicken alpha 7 receptors, but for other ligands there were large species-specific differences in binding affinity. These results paralleled properties of alpha 7 homomers expressed in Xenopus oocytes. Human alpha 7 homomers exhibited rapidly desensitizing, inwardly rectifying, agonist-induced, cation currents that triggered Ca(2+)-sensitive Cl- channels in the oocytes. A change in efficacy from partial agonist for chicken alpha 7 homomers to full agonist for human alpha 7 homomers was exhibited by 1,1-dimethyl-4-phenylpiperazinium. This result reveals a large species-specific pharmacological difference, despite small differences in alpha 7 sequences. This is important for understanding the effects of these drugs in humans and for identifying amino acids that may contribute to the acetylcholine binding site, for analysis by in vitro mutagenesis. These results also characterize properties of native alpha 7 receptors and alpha 7 homomers that will provide criteria for functional properties expected of structural subunits, when these can be identified, cloned, and coexpressed with alpha 7 subunits.

5',5'''-P1, P4 diadenosine tetraphosphate (Ap4A): a putative initiator of DNA replication.

PubMed

Baril, E F; Coughlin, S A; Zamecnik, P C

1985-01-01

The proposal that Ap4A acts as an inducer of DNA replication is based primarily on two pieces of evidence (7). The intracellular levels of Ap4A increase ten- to 1000-fold as cells progress into S phase and the introduction of Ap4A into nonproliferating cells stimulated DNA synthesis. There is also some additional suggestive evidence such as the binding of Ap4A to a protein that is associated with multiprotein forms of the replicative DNA polymerase alpha and the ability of this enzyme to use Ap4A as a primer for DNA synthesis in vitro with single-stranded DNA templates. These observations have stimulated interest in the cellular metabolism of Ap4A. This is well since there is a great need for additional experimentation in order to clearly establish Ap4A as an inducer of DNA replication. Microinjection experiments of Ap4A into quiescent cells are needed in order to ascertain if Ap4A will stimulate DNA replication and possibly cell division in intact cells. Studies of the effects of nonhydrolyzable analogs of Ap4A on DNA replication in intact quiescent cells could also prove valuable. Although Ap4A can function as a primer for in vitro DNA synthesis by DNA polymerase alpha this may not be relevant in regard to its in vivo role in DNA replication. Ap4A in vivo could interact with key protein(s) in DNA replication and in this way act as an effector molecule in the initiation of DNA replication. In this regard the interaction of Ap4A with a protein associated with a multiprotein form of DNA polymerase alpha isolated from S-phase cells is of interest. More experiments are required to determine if there is a specific target protein(s) for Ap4A in vivo and what its role in DNA replication is. The cofractionation of tryptophanyl-tRNA synthetase with the replicative DNA polymerase alpha from animal and plant cells is of interest. The DNA polymerase alpha from synchronized animal cells also interacted with Ap4A. Although the plant cell alpha-like DNA polymerase did not

Structural basis for DNA binding by replication initiator Mcm10

DOE Office of Scientific and Technical Information (OSTI.GOV)

Warren, Eric M.; Vaithiyalingam, Sivaraja; Haworth, Justin

2009-06-30

Mcm10 is an essential eukaryotic DNA replication protein required for assembly and progression of the replication fork. The highly conserved internal domain (Mcm10-ID) has been shown to physically interact with single-stranded (ss) DNA, DNA polymerase alpha, and proliferating cell nuclear antigen (PCNA). The crystal structure of Xenopus laevis Mcm10-ID presented here reveals a DNA binding architecture composed of an oligonucleotide/oligosaccharide-fold followed in tandem by a variant and highly basic zinc finger. NMR chemical shift perturbation and mutational studies of DNA binding activity in vitro reveal how Mcm10 uses this unique surface to engage ssDNA. Corresponding mutations in Saccharomyces cerevisiae resultmore » in increased sensitivity to replication stress, demonstrating the functional importance of DNA binding by this region of Mcm10 to replication. In addition, mapping Mcm10 mutations known to disrupt PCNA, polymerase alpha, and DNA interactions onto the crystal structure provides insight into how Mcm10 might coordinate protein and DNA binding within the replisome.« less

Complete primary structure of rainbow trout type I collagen consisting of alpha1(I)alpha2(I)alpha3(I) heterotrimers.

PubMed

Saito, M; Takenouchi, Y; Kunisaki, N; Kimura, S

2001-05-01

The subunit compositions of skin and muscle type I collagens from rainbow trout were found to be alpha1(I)alpha2(I)alpha3(I) and [alpha1(I)](2)alpha2(I), respectively. The occurrence of alpha3(I) has been observed only for bonyfish. The skin collagen exhibited more susceptibility to both heat denaturation and MMP-13 digestion than the muscle counterpart; the former had a lower denaturation temperature by about 0.5 degrees C than the latter. The lower stability of skin collagen, however, is not due to the low levels of imino acids because the contents of Pro and Hyp were almost constant in both collagens. On the other hand, some cDNAs coding for the N-terminal and/or a part of triple-helical domains of proalpha(I) chains were cloned from the cDNA library of rainbow trout fibroblasts. These cDNAs together with the previously cloned collagen cDNAs gave information about the complete primary structure of type I procollagen. The main triple-helical domain of each proalpha(I) chain had 338 uninterrupted Gly-X-Y triplets consisting of 1014 amino acids and was unique in its high content of Gly-Gly doublets. In particular, the bonyfish-specific alpha(I) chain, proalpha3(I) was characterized by the small number of Gly-Pro-Pro triplets, 19, and the large number of Gly-Gly doublets, 38, in the triple-helical domain, compared to 23 and 22, respectively, for proalpha1(I). The small number of Gly-Pro-Pro and the large number of Gly-Gly in proalpha3(I) was assumed to partially loosen the triple-helical structure of skin collagen, leading to the lower stability of skin collagen mentioned above. Finally, phylogenetic analyses revealed that proalpha3(I) had diverged from proalpha1(I). This study is the first report of the complete primary structure of fish type I procollagen.

Improved electro-transformation of highly DNA-restrictive corynebacteria with DNA extracted from starved Escherichia coli.

PubMed

Ankri, S; Reyes, O; Leblon, G

1996-07-01

Differences of up to 33 000-fold in electro-transformability of highly DNA restrictive corynebacteria are observed in the DNA of a shuttle plasmid extracted from Escherichia coli hosts propagated in different nutritional conditions. Growth of the host in minimal medium increases plasmid transformability, whereas growth on rich media decreases it. In the E. coli DH5 alpha host, the starvation-dependent increase DNA transformability is reverted by supplementing with methionine, an obligate 5-adenosyl-methionine (SAM) precursor. This suggests that an E. coli nutritionally modulated SAM-dependent DNA-methyltransferase may be involved in this phenomenon.

Association of Satellites with a Mastrevirus in Natural Infection: Complexity of Wheat Dwarf India Virus Disease

PubMed Central

Kumar, Jitendra; Kumar, Jitesh; Singh, Sudhir P.

2014-01-01

ABSTRACT In contrast to begomoviruses, mastreviruses have not previously been shown to interact with satellites. This study reports the first identification of the association of satellites with a mastrevirus in field-grown plants. Two alphasatellite species were detected in different field samples of wheat infected with Wheat Dwarf India Virus (WDIV), a Cotton leaf curl Multan alphasatellite (CLCuMA) and a Guar leaf curl alphasatellite (GLCuA). In addition to the alphasatellites, a betasatellite, Ageratum yellow leaf curl betasatellite (AYLCB), was also identified in the wheat samples. No begomovirus was detected in the wheat samples, thus establishing association of the above-named satellites with WDIV. Agrobacterium-mediated inoculation of WDIV in wheat, in the presence of either of the alphasatellites or the betasatellite, resulted in infections inducing more severe symptoms. WDIV efficiently maintained each of the alphasatellites and the betasatellite in wheat. The satellites enhanced the level of WDIV DNA in wheat. Inoculation of the satellites isolated from wheat with various begomoviruses into Nicotiana tabacum demonstrated that these remain capable of interacting with the viruses with which they were first identified. Virus-specific small RNAs accumulated in wheat upon infection with WDIV but were lower in abundance in plants coinfected with the satellites, suggesting that both the alphasatellites and the betasatellite suppress RNA silencing. These results suggest that the selective advantage for the maintenance of the alphasatellites and the betasatellite by WDIV in the field is in overcoming RNA silencing-mediated host defense. IMPORTANCE Wheat is the most widely cultivated cereal crop in the world. A number of viruses are important pathogens of wheat, including the viruses of the genus Mastrevirus, family Geminiviridae. This study reports the association of subgenomic components, called satellites (alpha- and betasatellites), with a mastrevirus, Wheat

Association of satellites with a mastrevirus in natural infection: complexity of Wheat dwarf India virus disease.

PubMed

Kumar, Jitendra; Kumar, Jitesh; Singh, Sudhir P; Tuli, Rakesh

2014-06-01

In contrast to begomoviruses, mastreviruses have not previously been shown to interact with satellites. This study reports the first identification of the association of satellites with a mastrevirus in field-grown plants. Two alphasatellite species were detected in different field samples of wheat infected with Wheat Dwarf India Virus (WDIV), a Cotton leaf curl Multan alphasatellite (CLCuMA) and a Guar leaf curl alphasatellite (GLCuA). In addition to the alphasatellites, a betasatellite, Ageratum yellow leaf curl betasatellite (AYLCB), was also identified in the wheat samples. No begomovirus was detected in the wheat samples, thus establishing association of the above-named satellites with WDIV. Agrobacterium-mediated inoculation of WDIV in wheat, in the presence of either of the alphasatellites or the betasatellite, resulted in infections inducing more severe symptoms. WDIV efficiently maintained each of the alphasatellites and the betasatellite in wheat. The satellites enhanced the level of WDIV DNA in wheat. Inoculation of the satellites isolated from wheat with various begomoviruses into Nicotiana tabacum demonstrated that these remain capable of interacting with the viruses with which they were first identified. Virus-specific small RNAs accumulated in wheat upon infection with WDIV but were lower in abundance in plants coinfected with the satellites, suggesting that both the alphasatellites and the betasatellite suppress RNA silencing. These results suggest that the selective advantage for the maintenance of the alphasatellites and the betasatellite by WDIV in the field is in overcoming RNA silencing-mediated host defense. Wheat is the most widely cultivated cereal crop in the world. A number of viruses are important pathogens of wheat, including the viruses of the genus Mastrevirus, family Geminiviridae. This study reports the association of subgenomic components, called satellites (alpha- and betasatellites), with a mastrevirus, Wheat Dwarf India Virus

Mutations in ATRX, encoding a SWI/SNF-like protein, cause diverse changes in the pattern of DNA methylation.

PubMed

Gibbons, R J; McDowell, T L; Raman, S; O'Rourke, D M; Garrick, D; Ayyub, H; Higgs, D R

2000-04-01

A goal of molecular genetics is to understand the relationship between basic nuclear processes, epigenetic changes and the numerous proteins that orchestrate these effects. One such protein, ATRX, contains a highly conserved plant homeodomain (PHD)-like domain, present in many chromatin-associated proteins, and a carboxy-terminal domain which identifies it as a member of the SNF2 family of helicase/ATPases. Mutations in ATRX give rise to characteristic developmental abnormalities including severe mental retardation, facial dysmorphism, urogenital abnormalities and alpha-thalassaemia. This circumstantial evidence suggests that ATRX may act as a transcriptional regulator through an effect on chromatin. We have recently shown that ATRX is localized to pericentromeric heterochromatin during interphase and mitosis, suggesting that ATRX might exert other chromatin-mediated effects in the nucleus. Moreover, at metaphase, some ATRX is localized at or close to the ribosomal DNA (rDNA) arrays on the short arms of human acrocentric chromosomes. Here we show that mutations in ATRX give rise to changes in the pattern of methylation of several highly repeated sequences including the rDNA arrays, a Y-specific satellite and subtelomeric repeats. Our findings provide a potential link between the processes of chromatin remodelling, DNA methylation and gene expression in mammalian development.

Lyman-alpha observations of comet Kohoutek 1973 XII with Copernicus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Drake, J.F.; Jenkins, E.B.; Bertaux, J.L.

1976-10-01

Comet Kohoutek 1973 XII was observed with the Princeton telescope-spectrometer on the Copernicus satellite on six occasions over a 1-month period starting on 1974 January 29. Positive detection of the cometary L..cap alpha.. emission profile was obtained on January 29 and February 2. Earlier observations of the geocoronal L..cap alpha.. emission profile allowed an instrumental intensity calibration and confirmation of the computed instrumental profile for an extended source at the L..cap alpha.. wavelength.After allowing for broadening by the instrument, we derived from the width of the L..cap alpha.. emission on January 29 a hydrogen-outflow velocity of 10.6 +- 1.8 kmmore » s/sup -1/. The intensity calibration combined with an appropriate cometary model led to cometary water-production rates with average values of 1.3 +- 0.4 x 10/sup 28/ molecules sr/sup -1/ s/sup -1/ for January 29 and 6.0 +- 2.5 x 10/sup 27/ molecules sr/sup -1/ s/sup -1/ for February 2. Only upper limits were obtained for L..cap alpha.. on and after February 14. Searches for OH and D led to negative results. (AIP)« less

Characterization of a photoproduct of 7,12-dimethylbenz[alpha]anthracene and its effects on chick-embryo cells in culture.

PubMed Central

Warshawsky, D; Kerns, E; Bissell, M J; Calvin, M

1977-01-01

A common impurity of 7,12-dimethylbenz[alpha]anthracene was more effective than 7,12-dimethylbenz[alpha]anthracene in inducing morphological alterations, and in causing an increase in glucose uptake, DNA synthesis and cell number in chick-embryo fibroblasts. Gradual morphological transformation follows the increase in DNA synthesis after 2 days when either primary or secondary cultures are treated with 3 microgram of the compound/ml. The compound, isolated from 7,12-dimethylbenz[alpha]anthracene by alumina column chromatography, was characterized by t.l.c., mass spectroscopy, carbon-hydrogen analysis, u.v. and nuclear-magnetic-resonance spectroscopy and thermal decomposition. It was the photo-oxidation product of 7,12-dimethylbenz[alpha]anthracene, 7,12-epidioxy-7,12-dimethylbenz[alpha]anthracene. It is suggested that some of the biological effects observed after treatment of cultures with 7,12-dimethylbenz[alpha]anthracene may be due in part to the presence of the photo-oxidation product. PMID:407902

Alpha-momorcharin: a ribosome-inactivating protein from Momordica charantia, possessing DNA cleavage properties.

PubMed

Wang, Shuzhen; Zheng, Yinzhen; Yan, Junjie; Zhu, Zhixuan; Wu, Zhihua; Ding, Yi

2013-11-01

Ribosome-inactivating proteins (RIPs) function to inhibit protein synthesis through the removal of specific adenine residues from eukaryotic ribosomal RNA and rending the 60S subunit unable to bind elongation factor 2. They have received much attention in biological and biomedical research due to their unique activities toward tumor cells, as well as the important roles in plant defense. Alpha-momorcharin (α-MC), a member of the type I family of RIPs, is rich in the seeds of Momordica charantia L. Previous studies demonstrated that α-MC is an effective antifungal and antibacterial protein. In this study, a detailed analysis of the DNase-like activity of α-MC was conducted. Results showed that the DNase-like activity toward plasmid DNA was time-dependent, temperature-related, and pH-stable. Moreover, a requirement for divalent metal ions in the catalytic domain of α-MC was confirmed. Additionally, Tyr(93) was found to be a critical residue for the DNase-like activity, while Tyr(134), Glu(183), Arg(186), and Trp(215) were activity-related residues. This study on the chemico-physical properties and mechanism of action of α-MC will improve its utilization in scientific research, as well as its potential industrial uses. These results may also assist in the characterization and elucidation of the DNase-like enzymatic properties of other RIPs.

Alpha-crystallins are involved in specific interactions with the murine gamma D/E/F-crystallin-encoding gene.

PubMed

Pietrowski, D; Durante, M J; Liebstein, A; Schmitt-John, T; Werner, T; Graw, J

1994-07-08

The promoter of the murine gamma E-crystallin (gamma E-Cry) encoding gene (gamma E-cry) was analyzed for specific interactions with lenticular proteins in a gel-retardation assay. A 21-bp fragment immediately downstream of the transcription initiation site (DOTIS) is demonstrated to be responsible for specific interactions with lens extracts. The DOTIS-binding protein(s) accept only the sense DNA strand as target; anti-sense or double-stranded DNA do not interact with these proteins. The DOTIS sequence element is highly conserved among the murine gamma D-, gamma E- and gamma F-cry and is present at comparable positions in the orthologous rat genes. Only a weak or even no protein-binding activity is observed if a few particular bases are changed, as in the rat gamma A-, gamma C- and gamma E-cry elements. DOTIS-binding proteins were found in commercially available bovine alpha-Cry preparations. The essential participation of alpha-Cry in the DNA-binding protein complex was confirmed using alpha-Cry-specific monoclonal antibody. The results reported here point to a novel function of alpha-Cry besides the structural properties in the lens.

T-cell receptor V sub. alpha. and C sub. alpha. alleles associated with multiple sclerosis and myasthenia gravis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oksenberg, J.R.; Cavalli-Sforza, L.L.; Steinman, L.

1989-02-01

Polymorphic markers in genes encoding the {alpha} chain of the human T-cell receptor (TcR) have been detected by Southern blot analysis in Pss I digests. Polymorphic bands were observed at 6.3 and 2.0 kilobases (kb) with frequencies of 0.30 and 0.44, respectively, in the general population. Using the polymerase chain reaction (PCR) method, the authors amplified selected sequences derived from the full-length TcR {alpha} cDNA probe. These PcR products were used as specific probes to demonstrate that the 6.3-kb polymorphic fragment hybridizes to the variable (V)-region probe and the 2.0-kb fragment hybridizes to the constant (C)-region probe. Segregation of themore » polymorphic bands was analyzed in family studies. To look for associations between these markers and autoimmune diseases, the authors have studied the restriction fragment length polymorphism distribution of the Pss I markers in patients with multiple sclerosis, myasthenia gravis, and Graves disease. Significant differences in the frequency of the polymorphic V{sub {alpha}} and C{sub {alpha}} markers were identified between patients and healthy individuals.« less

Measurements of geomagnetically trapped alpha particles, 1968-1970. I - Quiet time distributions

NASA Technical Reports Server (NTRS)

Krimigis, S. M.; Verzariu, P.

1973-01-01

Results of observations of geomagnetically trapped alpha particles over the energy range from 1.18 to 8 MeV performed with the aid of the Injun 5 polar-orbiting satellite during the period from September 1968 to May 1970. Following a presentation of a time history covering this entire period, a detailed analysis is made of the magnetically quiet period from Feb. 11 to 28, 1970. During this period the alpha particle fluxes and the intensity ratio of alpha particles to protons attained their lowest values in approximately 20 months; the alpha particle intensity versus L profile was most similar to the proton profile at the same energy per nucleon interval; the intensity ratio was nearly constant as a function of L in the same energy per nucleon representation, but rose sharply with L when computed in the same total energy interval; the variation of alpha particle intensity with B suggested a steep angular distribution at small equatorial pitch angles, while the intensity ratio showed little dependence on B; and the alpha particle spectral parameter showed a markedly different dependence on L from the equivalent one for protons.

TNF-alpha SNP haplotype frequencies in equidae.

PubMed

Brown, J J; Ollier, W E R; Thomson, W; Matthews, J B; Carter, S D; Binns, M; Pinchbeck, G; Clegg, P D

2006-05-01

Tumour necrosis factor alpha (TNF-alpha) is a pro-inflammatory cytokine that plays a crucial role in the regulation of inflammatory and immune responses. In all vertebrate species the genes encoding TNF-alpha are located within the major histocompatability complex. In the horse TNF-alpha has been ascribed a role in a variety of important disease processes. Previously two single nucleotide polymorphisms (SNPs) have been reported within the 5' un-translated region of the equine TNF-alpha gene. We have examined the equine TNF-alpha promoter region further for additional SNPs by analysing DNA from 131 horses (Equus caballus), 19 donkeys (E. asinus), 2 Grant's zebras (E. burchellii boehmi) and one onager (E. hemionus). Two further SNPs were identified at nucleotide positions 24 (T/G) and 452 (T/C) relative to the first nucleotide of the 522 bp polymerase chain reaction product. A sequence variant at position 51 was observed between equidae. SNaPSHOT genotyping assays for these and the two previously reported SNPs were performed on 457 horses comprising seven different breeds and 23 donkeys to determine the gene frequencies. SNP frequencies varied considerably between different horse breeds and also between the equine species. In total, nine different TNF-alpha promoter SNP haplotypes and their frequencies were established amongst the various equidae examined, with some haplotypes being found only in horses and others only in donkeys or zebras. The haplotype frequencies observed varied greatly between different horse breeds. Such haplotypes may relate to levels of TNF-alpha production and disease susceptibility and further investigation is required to identify associations between particular haplotypes and altered risk of disease.

alpha,beta-poly(asparthylhydrazide)-glycidyltrimethylammonium chloride copolymers (PAHy-GTA): novel polymers with potential for DNA delivery.

PubMed

Pedone, E; Cavallaro, G; Richardson, S C; Duncan, R; Giammona, G

2001-11-09

Hydrophilic polycations form complexes when mixed with plasmids. Following functionalisation with glycidyltrimethylammonium chloride (GTA) alpha,beta-poly(asparthylhydrazide) (PAHy), a water-soluble synthetic macromolecule, becomes polycationic and potentially useful for systemic gene delivery. Initially the biocompatibility of PAHy and PAHy-GTA derivatives with different degrees of positive charge substitution were studied and it was shown that PAHy-GTA was neither haemolytic nor cytotoxicity up to 1 mg/ml. After intravenous injection (125)I-labelled PAHy-GTA derivative containing 46 mol% (PAHy-GTA(b)) of trimethylammonium groups did not accumulate in the liver (4.1+/-0.9% of the recovered dose after 1 h) but was subjected to renal excretion (45+/-21% of the recovered dose was in the kidneys after 1 h). PAHy-GTA formed complexes with DNA (gel retardation) and they protected against degradation by DNase II. Finally the ability of the PAHy-GTA(b) derivative to mediate the transfection of HepG2 cells using the marker gene beta-galactosidase was studied. The optimum plasmid/polymer mass ratio was examined in comparison to LipofectACE, Lipofectin and polyethylenimine.

The p53 inhibitor, pifithrin-{alpha}, suppresses self-renewal of embryonic stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Abdelalim, Essam Mohamed, E-mail: essam_abdelalim@yahoo.com; Department of Cytology and Histology, Faculty of Veterinary Medicine, Suez Canal University, Ismailia 41522; Tooyama, Ikuo

2012-04-13

Highlights: Black-Right-Pointing-Pointer We determine the role of p53 in ES cells under unstressful conditions. Black-Right-Pointing-Pointer PFT-{alpha} suppresses ES cell proliferation. Black-Right-Pointing-Pointer PFT-{alpha} induces ES cell cycle arrest. Black-Right-Pointing-Pointer PFT-{alpha} downregulates Nanog and cyclin D1. -- Abstract: Recent studies have reported the role of p53 in suppressing the pluripotency of embryonic stem (ES) cells after DNA damage and blocking the reprogramming of somatic cells into induced pluripotent stem (iPS) cells. However, to date no evidence has been presented to support the function of p53 in unstressed ES cells. In this study, we investigated the effect of pifithrin (PFT)-{alpha}, an inhibitor ofmore » p53-dependent transcriptional activation, on self-renewal of ES cells. Our results revealed that treatment of ES cells with PFT-{alpha} resulted in the inhibition of ES cell propagation in a dose-dependent manner, as indicated by a marked reduction in the cell number and colony size. Also, PFT-{alpha} caused a cell cycle arrest and significant reduction in DNA synthesis. In addition, inhibition of p53 activity reduced the expression levels of cyclin D1 and Nanog. These findings indicate that p53 pathway in ES cells rather than acting as an inactive gene, is required for ES cell proliferation and self-renewal under unstressful conditions.« less

Characterization of the human pH- and PKA-activated ClC-2G(2 alpha) Cl- channel.

PubMed

Sherry, A M; Stroffekova, K; Knapp, L M; Kupert, E Y; Cuppoletti, J; Malinowska, D H

1997-08-01

A ClC-2G(2 alpha) Cl- channel was identified to be present in human lung and stomach, and a partial cDNA for this Cl- channel was cloned from a human fetal lung library. A full-length expressible human ClC-2G(2 alpha) cDNA was constructed by ligation of mutagenized expressible rabbit ClC-2G(2 alpha) cDNA with the human lung ClC-2G(2 alpha) cDNA, expressed in oocytes, and characterized at the single-channel level. Adenosine 3',5'-cyclic monophosphate-dependent protein kinase (PKA) treatment increased the probability of opening of the channel (Po). After PKA activation, the channel exhibited a linear (r = 0.99) current-voltage curve with a slope conductance of 22.1 +/- 0.8 pS in symmetric 800 mM tetraethylammonium chloride (TEACl; pH 7.4). Under fivefold gradient conditions of TEACl, a reversal potential of +21.5 +/- 2.8 mV was measured demonstrating anion-to-cation discrimination. As previously demonstrated for the rabbit ClC-2G(2 alpha) Cl- channel, the human analog, hClC-2G(2 alpha), was active at pH 7.4 as well as when the pH of the extracellular face of the channel (trans side of the bilayer; pHtrans) was asymmetrically reduced to pH 3.0. The extent of PKA activation was dependent on pHtrans. With PKA treatment, Po increased fourfold with a pHtrans of 7.4 and eightfold with a pHtrans of 3.0. Effects of sequential PKA addition followed by pHtrans reduction on the same channel suggested that the PKA- and pH-dependent increases in channel Po were separable and cumulative. Northern analysis showed ClC-2G(2 alpha) mRNA to be present in human adult and fetal lung and adult stomach, and quantitative reverse transcriptase-polymerase chain reaction showed this channel to be present in the adult human lung and stomach at about one-half the level found in fetal lung. The findings of the present study suggest that the ClC-2G(2 alpha) Cl- channel may play an important role in Cl- transport in the fetal and adult human lung.

Observation of Lyman-alpha emission in interplanetary space

NASA Technical Reports Server (NTRS)

Bertaux, J. L.; Blamont, J. E.

1972-01-01

The extraterrestrial Lyman-alpha emission was mapped by the OGO 5 satellite, when it was outside the geocorona. Three maps, obtained at different periods of the year, are presented and analyzed. The results suggest that at least half of the emission takes place in the solar system, and give strong support to the theory that in its motion toward the apex, the sun crosses neutral atomic hydrogen of interstellar origin, giving rise to an apparent interstellar wind.

Alpha-phellandrene-induced DNA damage and affect DNA repair protein expression in WEHI-3 murine leukemia cells in vitro.

PubMed

Lin, Jen-Jyh; Wu, Chih-Chung; Hsu, Shu-Chun; Weng, Shu-Wen; Ma, Yi-Shih; Huang, Yi-Ping; Lin, Jaung-Geng; Chung, Jing-Gung

2015-11-01

Although there are few reports regarding α-phellandrene (α-PA), a natural compound from Schinus molle L. essential oil, there is no report to show that α-PA induced DNA damage and affected DNA repair associated protein expression. Herein, we investigated the effects of α-PA on DNA damage and repair associated protein expression in murine leukemia cells. Flow cytometric assay was used to measure the effects of α-PA on total cell viability and the results indicated that α-PA induced cell death. Comet assay and 4,6-diamidino-2-phenylindole dihydrochloride staining were used for measuring DNA damage and condensation, respectively, and the results indicated that α-PA induced DNA damage and condensation in a concentration-dependent manner. DNA gel electrophoresis was used to examine the DNA damage and the results showed that α-PA induced DNA damage in WEHI-3 cells. Western blotting assay was used to measure the changes of DNA damage and repair associated protein expression and the results indicated that α-PA increased p-p53, p-H2A.X, 14-3-3-σ, and MDC1 protein expression but inhibited the protein of p53, MGMT, DNA-PK, and BRCA-1. © 2014 Wiley Periodicals, Inc.

Two views of the Andromeda Galaxy H-alpha and far infrared

NASA Technical Reports Server (NTRS)

Devereux, Nicholas A.; Price, Rob; Wells, Lisa A.; Duric, Neb

1994-01-01

A complete H-alpha image of the Andromeda Galaxy (M31) is presented allowing the first direct measurement of the total H-alpha luminosity which is (7.3 +/- 2.4) x 10(exp 6) solar luminosity. The H-alpha emission is associated with three morphologically distinct components; a large scale star-forming ring, approximately 1.65 deg in diameter, contributing 66% of the total H-alpha emission, a bright nucleus contributing 6% of the total H-alpha emission with the remaining 28% contributed by a previously unidentified component of extended and filamentary H-alpha emission interior to the star forming ring. The correspondence between the H-alpha image and the Infrared Astronomy Satellite (IRAS) far-infrared high resolution image is striking when both are convolved to a common resolution of 105 arcsec. The close correspondence between the far-infrared and H-alpha images suggests a common origin for the two emissions. The star-forming ring contributes 70% of the far-infrared luminosity of M31. Evidence that the ring emission is energized by high mass stars includes the fact that peaks in the far-infrared emission coincide identically with H II regions in the H-alpha image. In addition, the far-infrared to H-alpha luminosity ratio within the star-forming ring is similar to what one would expect for H II regions powered by stars of spectral types ranging between O9 and B0. The origin of the filamentary H-alpha and far-infrared luminosity interior to the star-forming ring is less clear, but it is almost certainly not produced by high mass stars.

In vitro DNA fragmentation of mitochondrial DNA caused by single-stranded breakage related to macroplasmodial senescence of the true slime mold, Physarum polycephalum.

PubMed

Abe, T; Takano, H; Sasaki, N; Mori, K; Kawano, S

2000-02-01

We found that mitochondrial DNA (mtDNA) isolated from Physarum polycephalum fragmented itself in weak ionic solutions. The mtDNA was dissolved in STE (saline Tris-EDTA: 150 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA), TE (10 mM Tris-HCl, 1 mM EDTA) and DW, and then electrophoresed in an agarose gel. The intact 86-kbp mtDNA band was seen in STE, but several novel bands appeared in TE and DW. In TE, two discrete bands appeared at 6.7-kbp (alpha-band) and 5.0-kbp (beta-band), whereas at least 17 discrete bands were observed in distilled water (DW). These fragmentation patterns were not stoichiometric, as seen when using restriction endonucleases, but were clearly different from the degradation of DNA caused by a physical shearing force or a contaminating nuclease. In this paper, we characterize this in vitro fragmentation of mtDNA from P. polycephalum. We located 19 fragments, including the alpha and beta fragments, on a mtDNA restriction map, and demonstrated that these cleavage sites were S1 nuclease-sensitive regions, which are single-stranded DNA regions such as nicks and gaps in the mtDNA. The alpha and beta fragments are derived from the region encoding ribosomal RNAs (rRNAs) and the ATP synthase (atpA) gene, while the other 17 fragments are not derived from any specific region, but the cleavage sites are located throughout the mtDNA molecule. In P. polycephalum, it is well known that the growth rate of macroplasmodia decreases with aging. Equal amounts of mtDNA from juvenile and aged macroplasmodia were electrophoresed and the frequency of the beta fragment in each sample was measured. The ratio of the beta band to the total signal including background was estimated to be 3.3-4.0% in juvenile macroplasmodia, whereas it increased to 8.3-28.2% in aged macroplasmodia. This result suggests that the in vitro fragmentation of mtDNA is associated with macroplasmodial senescence. The single-stranded breakage of mtDNA of P. polycephalum may accumulate with age.

Tetris Is a Foldback Transposon that Provided the Building Blocks for an Emerging Satellite DNA of Drosophila virilis

PubMed Central

Dias, Guilherme B.; Svartman, Marta; Delprat, Alejandra; Ruiz, Alfredo; Kuhn, Gustavo C.S.

2014-01-01

Transposable elements (TEs) and satellite DNAs (satDNAs) are abundant components of most eukaryotic genomes studied so far and their impact on evolution has been the focus of several studies. A number of studies linked TEs with satDNAs, but the nature of their evolutionary relationships remains unclear. During in silico analyses of the Drosophila virilis assembled genome, we found a novel DNA transposon we named Tetris based on its modular structure and diversity of rearranged forms. We aimed to characterize Tetris and investigate its role in generating satDNAs. Data mining and sequence analysis showed that Tetris is apparently nonautonomous, with a structure similar to foldback elements, and present in D. virilis and D. americana. Herein, we show that Tetris shares the final portions of its terminal inverted repeats (TIRs) with DAIBAM, a previously described miniature inverted transposable element implicated in the generation of chromosome inversions. Both elements are likely to be mobilized by the same autonomous TE. Tetris TIRs contain approximately 220-bp internal tandem repeats that we have named TIR-220. We also found TIR-220 repeats making up longer (kb-size) satDNA-like arrays. Using bioinformatic, phylogenetic and cytogenomic tools, we demonstrated that Tetris has contributed to shaping the genomes of D. virilis and D. americana, providing internal tandem repeats that served as building blocks for the amplification of satDNA arrays. The β-heterochromatic genomic environment seemed to have favored such amplification. Our results imply for the first time a role for foldback elements in generating satDNAs. PMID:24858539

Properties of nearby interstellar hydrogen deduced from Lyman-alpha sky background measurements

NASA Technical Reports Server (NTRS)

Thomas, G. E.

1972-01-01

For a sufficiently rapid relative motion of the solar system and the nearby interstellar gas, neutral atoms may be expected to penetrate the heliosphere before becoming ionized. Recent satellite measurements of the Lyman alpha emission above the geocorona indicate such an interstellar wind of neutral hydrogen emerging from the direction of Sagittarius and reaching to within a few astronomical units of the sun. A detailed model of the scattering of solar Lyman alpha from the spatial distribution of neutral hydrogen in interplanetary space is presented. This asymmetric distribution is established by solar wind and solar ultraviolet ionization processes along the trajectories of the incoming hydrogen atoms. The values of the interstellar density, the relative velocity, and the gas temperature are adjusted to agree with the Lyman alpha measurements. The results may be interpreted in terms of two models, the cold model and the hot model of the interstellar gas, depending on whether galactic Lyman alpha emission is present at its maximum allowable value or negligibly small.

The alpha subunit of the epithelial sodium channel in the mouse: developmental regulation of its expression.

PubMed

Dagenais, A; Kothary, R; Berthiaume, Y

1997-09-01

Sodium reabsorption by the amiloride-sensitive sodium channel of epithelial cells plays a crucial role in the management of ionic composition and fluid volume in the body. In the respiratory system, sodium transport is involved in the clearance of pulmonary edema and of liquid secreted during fetal life at birth. We have cloned a partial cDNA of the alpha subunit of the mouse amiloride-sensitive sodium channel (alpha mENaC). In the region of comparison, the mouse alpha subunit shows 92% identity at the DNA level and 95% identity at the amino acid level with the rat sequence. The kidneys, lungs, and distal colon are major sites of expression of a 3.5-kb alpha mENaC mRNA. During mouse development, alpha mENaC transcripts appear late during gestation (d 17.5) and are expressed continuously thereafter. In the distal colon, a short 1.2-kb mRNA deleted of the 5' part of the transcript is detected during gestation and is replaced gradually by the mature 3.5-kb transcript after birth. Alpha mENaC and alpha1 Na+-K+-ATPase mRNAs have an expression profile that is modulated similarly during development for a given tissue. The expression of alpha mENaC transcripts increases transiently in the lungs at birth (2.5-fold), as for alpha1 Na+-K+-ATPase mRNAs (1.5-fold), suggesting that the expression of several components of the sodium transport system is modulated in the lungs at that time. In the kidney, there is no significant increase of alpha mENaC and alpha1 Na+-K+-ATPase mRNAs in newborns.

DNA damage in spermatozoa from infertile men with varicocele evaluated by sperm chromatin dispersion and DBD-FISH.

PubMed

Cortés-Gutiérrez, Elva I; Dávila-Rodríguez, Martha I; Fernández, José Luis; López-Fernández, Carmen; Aragón-Tovar, Anel R; Urbina-Bernal, Luis C; Gosálvez, Jaime

2016-01-01

Evaluation of DNA integrity is an important test, possessing greater diagnostic and prognostic significance for couples requiring assisted reproduction. In this study, we evaluate the levels of DNA damage in infertile patients with varicocele with respect to fertile males by the sperm chromatin dispersion (SCD) test. The presence of DNA breaks in spermatozoa was confirmed by DNA breakage detection-fluorescence in situ hybridization (DBD-FISH). In this study, the frequency of sperm cells with fragmented DNA was studied in a group of 20 infertile patients with varicocele and compared with 20 fertile males. The spermatozoa were processed to classify different levels of DNA fragmentation using the Halosperm(®) kit, an improved SCD test, and DBD-FISH. Patients with varicocele showed 25.54 ± 28.17 % of spermatozoa with fragmented DNA, significantly higher than those of the group of fertile subjects (11.54 ± 3.88 %). The proportion of degraded cells in total sperm cells with fragmented DNA was sixfold higher in the case of patients with varicocele. The presence of DNA breaks in spermatozoa was confirmed by DBD-FISH. 5-bp Classical satellite-2 regions showed greater sensitivity to damage or "breakage" than alphoid satellite regions. Our finding preliminary demonstrated an increase of DNA fragmentation associated to severe sperm damage, in infertile patients with varicocele with respect to fertile males. 5-bp Classical satellite-2 regions showed greater sensitivity to damage or "breakage" than alphoid satellite regions.

DNA probe for lactobacillus delbrueckii

DOE Office of Scientific and Technical Information (OSTI.GOV)

Delley, M.; Mollet, B.; Hottinger, H.

1990-06-01

From a genomic DNA library of Lactobacillus delbrueckii subsp. bulgaricus, a clone was isolated which complements a leucine auxotrophy of an Escherichia coli strain (GE891). Subsequent analysis of the clone indicated that it could serve as a specific DNA probe. Dot-blot hybridizations with over 40 different Lactobacillus strains showed that this clone specifically recognized L. delbrueckii subsp. delbrueckii, bulgaricus, and lactis. The sensitivity of the method was tested by using an {alpha}-{sup 32}P-labeled probe.

Sequences Associated with Centromere Competency in the Human Genome

PubMed Central

Hayden, Karen E.; Strome, Erin D.; Merrett, Stephanie L.; Lee, Hye-Ran; Rudd, M. Katharine

2013-01-01

Centromeres, the sites of spindle attachment during mitosis and meiosis, are located in specific positions in the human genome, normally coincident with diverse subsets of alpha satellite DNA. While there is strong evidence supporting the association of some subfamilies of alpha satellite with centromere function, the basis for establishing whether a given alpha satellite sequence is or is not designated a functional centromere is unknown, and attempts to understand the role of particular sequence features in establishing centromere identity have been limited by the near identity and repetitive nature of satellite sequences. Utilizing a broadly applicable experimental approach to test sequence competency for centromere specification, we have carried out a genomic and epigenetic functional analysis of endogenous human centromere sequences available in the current human genome assembly. The data support a model in which functionally competent sequences confer an opportunity for centromere specification, integrating genomic and epigenetic signals and promoting the concept of context-dependent centromere inheritance. PMID:23230266

Coordination of the Ser2056 and Thr2609 Clusters of DNA-PKcs in Regulating Gamma Rays and Extremely Low Fluencies of Alpha-Particle Irradiation to G0/G1 Phase Cells.

PubMed

Nagasawa, Hatsumi; Lin, Yu-Fen; Kato, Takamitsu A; Brogan, John R; Shih, Hung-Ying; Kurimasa, Akihiro; Bedford, Joel S; Chen, Benjamin P C; Little, John B

2017-02-01

The catalytic subunit of DNA dependent protein kinase (DNA-PKcs) and its kinase activity are critical for mediation of non-homologous end-joining (NHEJ) of DNA double-strand breaks (DSB) in mammalian cells after gamma-ray irradiation. Additionally, DNA-PKcs phosphorylations at the T2609 cluster and the S2056 cluster also affect DSB repair and cellular sensitivity to gamma radiation. Previously we reported that phosphorylations within these two regions affect not only NHEJ but also homologous recombination repair (HRR) dependent DSB repair. In this study, we further examine phenotypic effects on cells bearing various combinations of mutations within either or both regions. Effects studied included cell killing as well as chromosomal aberration induction after 0.5-8 Gy gamma-ray irradiation delivered to synchronized cells during the G 0 /G 1 phase of the cell cycle. Blocking phosphorylation within the T2609 cluster was most critical regarding sensitization and depended on the number of available phosphorylation sites. It was also especially interesting that only one substitution of alanine in each of the two clusters separately abolished the restoration of wild-type sensitivity by DNA-PKcs. Similar patterns were seen for induction of chromosomal aberrations, reflecting their connection to cell killing. To study possible change in coordination between HRR and NHEJ directed repair in these DNA-PKcs mutant cell lines, we compared the induction of sister chromatid exchanges (SCEs) by very low fluencies of alpha particles with mutant cells defective in the HRR pathway that is required for induction of SCEs. Levels of true SCEs induced by very low fluence of alpha-particle irradiation normally seen in wild-type cells were only slightly decreased in the S2056 cluster mutants, but were completely abolished in the T2609 cluster mutants and were indistinguishable from levels seen in HRR deficient cells. Again, a single substitution in the S2056 together with a single

Investigation of atomic oxygen-surface interactions related to measurements with dual air density explorer satellites

NASA Technical Reports Server (NTRS)

Wood, B. J.; Ablow, C. M.; Wise, H.

1973-01-01

For a number of candidate materials of construction for the dual air density explorer satellites the rate of oxygen atom loss by adsorption, surface reaction, and recombination was determined as a function of surface and temperature. Plain aluminum and anodized aluminum surfaces exhibit a collisional atom loss probability alpha .01 in the temperature range 140 - 360 K, and an initial sticking probability. For SiO coated aluminum in the same temperature range, alpha .001 and So .001. Atom-loss on gold is relatively rapid alpha .01. The So for gold varies between 0.25 and unity in the temperature range 360 - 140 K.

Analysis of Ag/FEP Thermal Control Blanket Performance From Multiple Satellites

NASA Technical Reports Server (NTRS)

Pippin, Gary; Normand, E.; Woll, S.; Munafo, Paul (Technical Monitor)

2001-01-01

Materials performance data from operational spacecraft and spacecraft experiments has slowly accumulated over the years. Much of this data is obtained indirectly by analyzing temperature measurements telemetered to ground. Due to the Space Transportation System (STS) program, materials performance data has also been obtained by flying materials on satellites, or on the Space Shuttle itself, for limited periods of time, and then returning the materials to ground for post-flight examination. During the course of NASA contract NAS8-98213, the authors have extended the analysis of data reported for selected materials flown on a variety of spacecraft over the past 30 years. In this paper we discuss changes in two properties of silver- or aluminum-backed (metallized) FEP Teflon due to exposure to specific environmental factors on-orbit. First, the change in solar absorptance (alpha(sub s)) of metallized FEP Teflon as a function of particulate radiation will be discussed. Data providing the change in solar absorptance of metallized FEP as a function of days on orbit has been published for NTS-2 satellite, several NavStar Global Positioning Satellites (GPS), the SCATHA experiment, and the ML-101 satellite. The unique approach that we have taken is to calculate the absorbed radiation dose within the Ag/FEP for the various satellite orbits, and to plot the change in solar absorptance, alpha(sub s), as a function of the dose; the behavior agrees with the same data from laboratory tests. Second, the variation of the atomic oxygen induced material recession rate of metallized FEP will be discussed. Data from certain Space Shuttle flights, a Lockheed experiment [approximately 105 days in low Earth orbit (LEO)], and the Long Duration Exposure Facility allows the variation of the material recession rate of Ag/FEP to be estimated under conditions that include atomic oxygen exposure.

Characterization of a novel isoform of alpha-nascent polypeptide-associated complex as IgE-defined autoantigen.

PubMed

Mossabeb, Roschanak; Seiberler, Susanne; Mittermann, Irene; Reininger, Renate; Spitzauer, Susanne; Natter, Susanne; Verdino, Petra; Keller, Walter; Kraft, Dietrich; Valenta, Rudolf

2002-10-01

The nascent polypeptide-associated complex is required for intracellular translocation of newly synthesized polypeptides in eukaryotic cells. It may also act as a transcriptional coactivator in humans and various eukaryotic organisms and binds to nucleic acids. Recently, we provided evidence that a component of nascent polypeptide-associated complex, alpha-nascent polypeptide-associated complex, represents an IgE-reactive autoantigen for atopic dermatitis patients. By oligonucleotide screening we isolated a complete cDNA coding for a so far unknown alpha-nascent polypeptide-associated complex isoform from a human epithelial cDNA library. Southern blot hybridization experiments provided further evidence that alpha-nascent polypeptide-associated complex is encoded by a gene family. Recombinant alpha-nascent polypeptide-associated complex was expressed in Escherichia coli as a soluble, His-tagged protein, and purified via nickel affinity chromatography. By circular dichroism analysis it is demonstrated that purified recombinant alpha-nascent polypeptide-associated complex represents a folded protein of mixed alpha-helical and beta-sheet conformation with unusual high thermal stability and remarkable refolding capacity. Complete recombinant alpha-nascent polypeptide-associated complex (215 amino acids) and its 86 amino acid C-terminal fragment specifically bound IgE autoantibodies. Recombinant alpha-nascent polypeptide-associated complex also inhibited IgE binding to natural alpha-nascent polypeptide-associated complex, demonstrating the presence of common IgE epitopes between the recombinant and natural protein. Furthermore, recombinant alpha-nascent polypeptide-associated complex induced specific lymphoproliferative responses in peripheral blood mononuclear cells of a sensitized atopic dermatitis patient. As has been proposed for environmental allergens it is possible that T cell responses to IgE-defined autoantigens may contribute to the chronic skin manifestations

Cloning, sequence, and expression of a blood group B active recombinant alpha-D-galactosidase from pinto bean (Phaseolus vulgaris).

PubMed

Davis, M O; Hata, D J; Johnson, S A; Jones, D E; Harmata, M A; Evans, M L; Walker, J C; Smith, D S

1997-07-01

A cDNA encoding pinto bean alpha-D-galactosidase [E.C. 3.2.1.22] was obtained by amplification of cDNA using highly conserved sequences found in eucaryotic alpha-D-galactosidases. Subsequently a full length Phaseolus cDNA clone was obtained that is 1537 nt long and contains untranslated 5' and 3' sequences. The nucleotide sequence of the cDNA has a high degree of homology with other eucaryotic alpha-D-galactosidase genes. The recombinant alpha-D-galactosidase (rGal) was expressed in Escherichia coli and purified by ion exchange and affinity chromatography. Purified rGal was homogeneous by SDS-PAGE and had relative masses of 40.1 and 45.4 kDa under nonreducing and reducing conditions, respectively. The N-terminal sequence of the expressed protein contained the sequence GNGLGQTPPMG corresponding to that deduced from the cDNA sequence. The native molecular weight for rGal was determined to be 32.18 kDa by Sephacryl S-200 chromatography. The specific activity of the rGal was 349 mu moles of PNP-alpha-D-galactopyranoside hydrolyzed per mg of pure rGal per min. rGal was highly specific for alpha-D-galactosyl residues and degraded B oligosaccharide. No detectable hemagglutinin or protease activity was present in the preparations. Furthermore, rGal was active against the blood group B antigen on native human erythrocytes in cell suspension assays. The only detectable RBC phenotypic change was loss of the B and P1 epitopes. Recombinant Phaseolus vulgaris alpha-D-galactosidase may have useful biotechnical applications in the potential mass production of enzymatically converted, universally transfusable type O RBCs. alpha-D-galactosidase [E.C. 3.2.1.22] has been purified from a variety of procaryotic and eucaryotic species. Most alpha-D-galactosidases have similar low molecular weight substrate specificities, but activity against high molecular weight substrates is variable. Terminal alpha-D-galactoside residues are present in glycoproteins and glycolipids. Some alpha

Scaling in nature: from DNA through heartbeats to weather

NASA Technical Reports Server (NTRS)

Havlin, S.; Buldyrev, S. V.; Bunde, A.; Goldberger, A. L.; Peng, C. K.; Stanley, H. E.

1999-01-01

The purpose of this report is to describe some recent progress in applying scaling concepts to various systems in nature. We review several systems characterized by scaling laws such as DNA sequences, heartbeat rates and weather variations. We discuss the finding that the exponent alpha quantifying the scaling in DNA in smaller for coding than for noncoding sequences. We also discuss the application of fractal scaling analysis to the dynamics of heartbeat regulation, and report the recent finding that the scaling exponent alpha is smaller during sleep periods compared to wake periods. We also discuss the recent findings that suggest a universal scaling exponent characterizing the weather fluctuations.

More efficient repair of DNA double-strand breaks in skeletal muscle stem cells compared to their committed progeny.

PubMed

Vahidi Ferdousi, Leyla; Rocheteau, Pierre; Chayot, Romain; Montagne, Benjamin; Chaker, Zayna; Flamant, Patricia; Tajbakhsh, Shahragim; Ricchetti, Miria

2014-11-01

The loss of genome integrity in adult stem cells results in accelerated tissue aging and is possibly cancerogenic. Adult stem cells in different tissues appear to react robustly to DNA damage. We report that adult skeletal stem (satellite) cells do not primarily respond to radiation-induced DNA double-strand breaks (DSBs) via differentiation and exhibit less apoptosis compared to other myogenic cells. Satellite cells repair these DNA lesions more efficiently than their committed progeny. Importantly, non-proliferating satellite cells and post-mitotic nuclei in the fiber exhibit dramatically distinct repair efficiencies. Altogether, reduction of the repair capacity appears to be more a function of differentiation than of the proliferation status of the muscle cell. Notably, satellite cells retain a high efficiency of DSB repair also when isolated from the natural niche. Finally, we show that repair of DSB substrates is not only very efficient but, surprisingly, also very accurate in satellite cells and that accurate repair depends on the key non-homologous end-joining factor DNA-PKcs. Copyright © 2014. Published by Elsevier B.V.

Improvement of heterologous protein production in Aspergillus oryzae by RNA interference with alpha-amylase genes.

PubMed

Nemoto, Takashi; Maruyama, Jun-ichi; Kitamoto, Katsuhiko

2009-11-01

Aspergillus oryzae RIB40 has three alpha-amylase genes (amyA, amyB, and amyC), and secretes alpha-amylase abundantly. However, large amounts of endogenous secretory proteins such as alpha-amylase can compete with heterologous protein in the secretory pathway and decrease its production yields. In this study, we examined the effects of suppression of alpha-amylase on heterologous protein production in A. oryzae, using the bovine chymosin (CHY) as a reporter heterologous protein. The three alpha-amylase genes in A. oryzae have nearly identical DNA sequences from those promoters to the coding regions. Hence we performed silencing of alpha-amylase genes by RNA interference (RNAi) in the A. oryzae CHY producing strain. The silenced strains exhibited a reduction in alpha-amylase activity and an increase in CHY production in the culture medium. This result suggests that suppression of alpha-amylase is effective in heterologous protein production in A. oryzae.

Molecular cloning of rock bream (Oplegnathus fasciatus) tumor necrosis factor-alpha and its effect on the respiratory burst activity of phagocytes.

PubMed

Kim, Min Sun; Hwang, Yoon Jung; Yoon, Ki Joon; Zenke, Kosuke; Nam, Yoon Kwon; Kim, Sung Koo; Kim, Ki Hong

2009-11-01

Rock bream (Oplegnathus fasciatus) tumor necrosis factor-alpha (rbTNF-alpha) gene was cloned, recombinantly produced, and the effect of the recombinant rbTNF-alpha on the respiratory burst activity of rock bream phagocytes was analyzed. Structurally, genomic DNA of rbTNF-alpha was comprised with four exons and three introns, and deduced amino acid sequence of its cDNA possessed the TNF family signature, a transmembrane domain, a protease cleavage site, and two cysteine residues, which are the typical characteristics of TNF-alpha gene in mammals and fish. The chemiluminescent (CL) response of rock bream phagocytes was significantly enhanced by pre-incubation with recombinant rbTNF-alpha, when opsonized zymosan was used as a stimulant of the respiratory burst. However, CL enhancing effect of the recombinant rbTNF-alpha was very weak when the respiratory burst activity of phagocytes was triggered with phorbol-12-myristate-13-acetate (PMA) instead of zymosan. These results suggest that rock bream TNF-alpha might have an ability to prime the respiratory burst activity of phagocytes against receptor-mediated phagocytosis inducing stimulants, such as zymosan, but have little ability against stimulants not accompanying receptor-mediated phagocytosis.

Genotyping of alpha-thalassemia deletions using multiplex polymerase chain reactions and gold nanoparticle-filled capillary electrophoresis.

PubMed

Chen, Yen-Ling; Shih, Chi-Jen; Ferrance, Jerome; Chang, Ya-Sian; Chang, Jan-Gowth; Wu, Shou-Mei

2009-02-13

A gold nanoparticle-filled capillary electrophoresis method combined with three multiplex polymerase chain reactions (PCRs) was established for simultaneous diagnosis of five common alpha-thalassemia deletions, including the -alpha(3.7) deletion, -alpha(4.2) deletion, Southeast Asian (--(SEA)), Filipino (--(FIL)) and Thai (--(THAI)) deletions. Gold nanoparticles (GNPs) were used as a pseudostationary phase to improve the resolution between DNA fragments in a low-viscosity polymer. To achieve the best CE separation, several parameters were evaluated for optimizing the separation conditions, including the capillary coating, the concentrations of polymer sieving matrix, the sizes and concentrations of GNPs, the buffer concentrations, and the pH. The final CE method for separating a 200-base pair (bp) DNA ladder and alpha-thalassemia deletions used a DB-17 capillary, 0.6% poly(ethylene oxide) (PEO) prepared in a mixture of GNP(32nm) solution and glycine buffer (25mM, pH 9.0) (80:20, v/v) as the sieving matrix with 1microM YO-PRO-1 for fluorescence detection; the applied voltage was -10kV (detector at anode side) and the separation temperature was 25 degrees C. Under these optimal conditions, 15 DNA fragments with sizes ranging from 0.2kb to 3.0kb were resolved within 11.5min. The RSDs of migration times were less than 2.81%. A total of 21 patients with alpha-thalassemia deletions were analyzed using this method, and all results showed good agreement with those obtained by gel electrophoresis.

Dynamic evolution of recurrent mass ejections observed in H-alpha and C IV lines

NASA Technical Reports Server (NTRS)

Schmieder, B.; Mein, P.; Martres, M. J.; Tandberg-Hanssen, E.

1984-01-01

The mass ejections of 1 September, 1980 are studied from observations obtained with the MSDP spectrograph and with the Ultraviolet Spectrometer and Polarimeter aboard the Solar Maximum Mission satellite. The analysis is focused on observations in the chromospheric H-alpha line and the transition region C IV 1548 A line. It is noted that cold and hot material had the same projection, although the upward C IV velocity structure was more extended than the H-alpha one. It is shown that the observed contrast of the H-alpha absorbing structure can be interpreted in terms of a dynamic cloud model overlying the chromosphere. Radial velocities of 25-30 km/s and -40 km/s are estimated for the first and second phases of ejection, respectively.

Defects in maintenance of mitochondrial DNA are associated with intramitochondrial nucleotide imbalances.

PubMed

Ashley, Neil; Adams, Susan; Slama, Abdelhamid; Zeviani, Massimo; Suomalainen, Anu; Andreu, Antonio L; Naviaux, Robert K; Poulton, Joanna

2007-06-15

Defects in mtDNA maintenance range from fatal multisystem childhood diseases, such as Alpers syndrome, to milder diseases in adults, including mtDNA depletion syndromes (MDS) and familial progressive external ophthalmoplegia (AdPEO). Most are associated with defects in genes involved in mitochondrial deoxynucleotide metabolism or utilization, such as mutations in thymidine kinase 2 (TK2) as well as the mtDNA replicative helicase, Twinkle and gamma polymerase (POLG). We have developed an in vitro system to measure incorporation of radiolabelled dNTPs into mitochondria of saponin permeabilized cells. We used this to compare the rates of mtDNA synthesis in cells from 12 patients with diseases of mtDNA maintenance. We observed reduced incorporation of exogenous alpha (32)P-dTTP in fibroblasts from a patient with Alpers syndrome associated with the A467T substitution in POLG, a patient with dGK mutations, and a patient with mtDNA depletion of unknown origin compared to controls. However, incorporation of alpha (32)P-dTTP relative to either cell doubling time or alpha (32)P-dCTP incorporation was increased in patients with thymidine kinase deficiency or PEO as the result of TWINKLE mutations compared with controls. The specific activity of newly synthesized mtDNA depends on the size of the endogenous pool diluting the exogenous labelled nucleotide. Our result is consistent with a deficiency in the intramitochondrial pool of dTTP relative to dCTP in cells from patients with TK2 deficiency and TWINKLE mutations. Such DNA precursor asymmetry could cause pausing of the replication complex and hence exacerbate the propensity for age-related mtDNA mutations. Because deviations from the normal concentrations of dNTPs are known to be mutagenic, we suggest that intramitochondrial nucleotide imbalance could underlie the multiple mtDNA mutations observed in these patients.

Mechanism of repression of the inhibin alpha-subunit gene by inducible 3',5'-cyclic adenosine monophosphate early repressor.

PubMed

Burkart, Anna D; Mukherjee, Abir; Mayo, Kelly E

2006-03-01

The rodent ovary is regulated throughout the reproductive cycle to maintain normal cyclicity. Ovarian follicular development is controlled by changes in gene expression in response to the gonadotropins FSH and LH. The inhibin alpha-subunit gene belongs to a group of genes that is positively regulated by FSH and negatively regulated by LH. Previous studies established an important role for inducible cAMP early repressor (ICER) in repression of alpha-inhibin. These current studies investigate the mechanisms of repression by ICER. It is not clear whether all four ICER isoforms expressed in the ovary can act as repressors of the inhibin alpha-subunit gene. EMSAs demonstrate binding of all isoforms to the inhibin alpha-subunit CRE (cAMP response element), and transfection studies demonstrate that all isoforms can repress the inhibin alpha-subunit gene. Repression by ICER is dependent on its binding to DNA as demonstrated by mutations to ICER's DNA-binding domain. These mutational studies also demonstrate that repression by ICER is not dependent on heterodimerization with CREB (CRE-binding protein). Competitive EMSAs show that ICER effectively competes with CREB for binding to the inhibin alpha CRE in vitro. Chromatin immunoprecipitation assays demonstrate a replacement of CREB dimers bound to the inhibin alpha CRE by ICER dimers in ovarian granulosa cells in response to LH signaling. Thus, there is a temporal association of transcription factors bound to the inhibin alpha-CRE controlling inhibin alpha-subunit gene expression.

Tetris is a foldback transposon that provided the building blocks for an emerging satellite DNA of Drosophila virilis.

PubMed

Dias, Guilherme B; Svartman, Marta; Delprat, Alejandra; Ruiz, Alfredo; Kuhn, Gustavo C S

2014-05-24

Transposable elements (TEs) and satellite DNAs (satDNAs) are abundant components of most eukaryotic genomes studied so far and their impact on evolution has been the focus of several studies. A number of studies linked TEs with satDNAs, but the nature of their evolutionary relationships remains unclear. During in silico analyses of the Drosophila virilis assembled genome, we found a novel DNA transposon we named Tetris based on its modular structure and diversity of rearranged forms. We aimed to characterize Tetris and investigate its role in generating satDNAs. Data mining and sequence analysis showed that Tetris is apparently nonautonomous, with a structure similar to foldback elements, and present in D. virilis and D. americana. Herein, we show that Tetris shares the final portions of its terminal inverted repeats (TIRs) with DAIBAM, a previously described miniature inverted transposable element implicated in the generation of chromosome inversions. Both elements are likely to be mobilized by the same autonomous TE. Tetris TIRs contain approximately 220-bp internal tandem repeats that we have named TIR-220. We also found TIR-220 repeats making up longer (kb-size) satDNA-like arrays. Using bioinformatic, phylogenetic and cytogenomic tools, we demonstrated that Tetris has contributed to shaping the genomes of D. virilis and D. americana, providing internal tandem repeats that served as building blocks for the amplification of satDNA arrays. The β-heterochromatic genomic environment seemed to have favored such amplification. Our results imply for the first time a role for foldback elements in generating satDNAs. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Spectroscopic Characterization of GEO Satellites with Gunma LOW Resolution Spectrograph

NASA Astrophysics Data System (ADS)

Endo, T.; Ono, H.; Hosokawa, M.; Ando, T.; Takanezawa, T.; Hashimoto, O.

The spectroscopic observation is potentially a powerful tool for understanding the Geostationary Earth Orbit (GEO) objects. We present here the results of an investigation of energy spectra of GEO satellites obtained from a groundbased optical telescope. The spectroscopic observations were made from April to June 2016 with the Gunma LOW resolution Spectrograph and imager (GLOWS) at the Gunma Astronomical Observatory (GAO) in JAPAN. The observation targets consist of eleven different satellites: two weather satellites, four communications satellites, and five broadcasting satellites. All the spectra of those GEO satellites are inferred to be solar-like. A number of well-known absorption features such as H-alpha, H-beta, Na-D,water vapor and oxygen molecules are clearly seen in thewavelength range of 4,000 - 8,000 Å. For comparison, we calculated the intensity ratio of the spectra of GEO satellites to that of the Moon which is the natural satellite of the earth. As a result, the following characteristics were obtained. 1) Some variations are seen in the strength of absorption features of water vapor and oxygen originated by the telluric atmosphere, but any other characteristic absorption features were not found. 2) For all observed satellites, the intensity ratio of the spectrum of GEO satellites decrease as a function of wavelength or to be flat. It means that the spectral reflectance of satellite materials is bluer than that of the Moon. 3) A characteristic dip at around 4,800 Å is found in all observed spectra of a weather satellite. Based on these observations, it is indicated that the characteristics of the spectrum are mainly derived from the solar panels because the apparent area of the solar cell is probably larger than that of the satellite body.

Prevalence of -alpha(3.7) and alpha alpha alpha(anti3.7) alleles in sickle cell trait and beta-thalassemia patients in Mexico.

PubMed

Nava, María Paulina; Ibarra, Bertha; Magaña, María Teresa; de la Luz Chávez, María; Perea, F Javier

2006-01-01

The aim of this study was to determine the frequency of alpha-globin gene mutations in three groups of Mexican unrelated individuals. The first two groups were normal and sickle cell trait individuals from the Costa Chica region, a place with a 12.8% frequency of HbS carriers, and the third group comprised of Mexican mestizo patients with beta-thalassemia. We searched for -alpha(3.7) and -alpha(4.2) alpha(+)-thalassemia deletion alleles, as well as the alpha alpha alpha(anti3.7) triplication through long-gap PCR. The alleles -alpha(3.7) and alpha alpha alpha(anti3.7) were found in the heterozygote state only; 19% of the normal subjects had the -alpha(3.7) allele, and 2% showed the alpha alpha alpha(anti3.7) allele. In individuals with the sickle cell trait, 17% had the -alpha(3.7) deletion, and the alpha alpha alpha(anti3.7) triplication was observed in 3% of these individuals. We revealed that 16% of the subjects with beta-thalassemia showed the -alpha(3.7) deletion and 28% the alpha alpha alpha(anti3.7) triplication. The -alpha(4.2) deletion was not detected in any individual. The frequency of the -alpha(3.7) allele was roughly the same in the three groups studied; this can be explained by the fact that the three groups have common genes from Africa and the Mediterranean, where a high prevalence of alpha(+)-thalassemia has been observed. To our knowledge, the frequency of alpha alpha alpha(anti3.7) triplication observed in the Mexican beta-thalassemia patients is the highest reported. As the -alpha(3.7) and alpha alpha alpha(anti3.7) alleles are very common in our selected populations, we believe that there is a need to investigate systematically the alpha-globin gene mutations in all hemoglobinopathies in the Mexican population.

Genomic in situ hybridization in interspecific hybrids of scallops (Bivalvia, Pectinidae) and localization of the satellite DNA Cf303, and the vertebrate telomeric sequences (TTAGGG)n on chromosomes of scallop Chlamys farreri (Jones & Preston, 1904).

PubMed

Hu, Liping; Jiang, Liming; Bi, Ke; Liao, Huan; Yang, Zujing; Huang, Xiaoting; Bao, Zhenmin

2018-01-01

Mitotic chromosome preparations of the interspecific hybrids Chlamys farreri (Jones & Preston, 1904) × Patinopecten yessoensis (Jay, 1857), C. farreri × Argopecten irradians (Lamarck, 1819) and C. farreri × Mimachlamys nobilis (Reeve, 1852) were used to compare two different scallop genomes in a single slide. Although genomic in situ hybridization (GISH) using genomic DNA from each scallop species as probe painted mitotic chromosomes of the interspecific hybrids, the painting results were not uniform; instead it showed species-specific distribution patterns of fluorescent signals among the chromosomes. The most prominent GISH-bands were mainly located at centromeric or telomeric regions of scallop chromosomes. In order to illustrate the sequence constitution of the GISH-bands, the satellite Cf303 sequences of C. farreri and the vertebrate telomeric (TTAGGG) n sequences were used to map mitotic chromosomes of C. farreri by fluorescence in situ hybridization (FISH). The results indicated that the GISH-banding pattern presented by the chromosomes of C. farreri is mainly due to the distribution of the satellite Cf303 DNA, therefore suggesting that the GISH-banding patterns found in the other three scallops could also be the result of the chromosomal distribution of other species-specific satellite DNAs.

Base-Pairing Systems Related to TNA: alpha-Threofuranosyl Oligonucleotides Containing Phosphoramidate Linkages

NASA Technical Reports Server (NTRS)

Meyer, Michael (Technical Monitor); Wu, Xiaolin; Guntha, Sreenivasulu; Ferenclc, Mathias; Krishnamurthy, Ramanarayanan; Eschenmoser, Albert

2002-01-01

(3'NH)- and (2'NH)-TNA, two isomeric phosphoramidate analogues of TNA (alpha-threofuranosyl-(3'-2') oligonucleotides), are shown to be efficient Watson-Crick base-pairing systems and to undergo intersystem crosspairing with TNA, RNA, and DNA.

The expression of a naturally occuriing truncated allele of an alpha-SNAP gene suppresses plant parasitic nematode infection

USDA-ARS?s Scientific Manuscript database

rhg1, defined within a 67 kb region of DNA on chromosome 18, is a major quantitative trait locus (QTL) in Glycine max (soybean) providing defense to the soybean cyst nematode (Heterodera glycines). Transcriptional mapping experiments identified an alpha soluble NSF attachment protein (alpha-SNAP) wi...

Local expression of interferon-alpha and interferon receptors in cervical intraepithelial neoplasia.

PubMed

Tirone, Nelson R; Peghini, Bethanea C; Barcelos, Ana Cristina M; Murta, Eddie F C; Michelin, Marcia A

2009-12-01

The present study evaluated mRNA expression of interferon-alpha (IFN-alpha), IFN-alpha receptor subunits (IFNAR-1 and IFNAR-2) and an IFN-stimulated gene encoding the enzyme 2',5'-oligoadenylate synthetase (2'5'OAS) in biopsies on patients with varying grades of cervical intraepithelial neoplasia (CIN I, II and III). Uterine cervix biopsies were collected from women with CIN I, II and III (n = 28) and controls without CIN lesions or human papilloma virus (HPV) infection (n = 17). The presence of high and low-risk HPV DNA was determined using hybrid capture. The mRNA levels of IFNAR-1, IFNAR-2, IFN-alpha and 2'5'OAS were determined by RT-PCR with specific primers. The control group exhibited a greater frequency of IFNAR-1 expression (10/17; 58.3%) than the CIN samples (4/28; 14.2%) (P = 0.0018), while, the expression of IFNAR-2 was also greater in the control samples (11/17; 64.7%) than in the patients with lesions (2/28; 7.1%) (P = 0.0018). Importantly, simultaneous expression of both receptors was observed only in the control group (8/17; 47.0%) (P = 0.0001). Among the CIN samples, there was one case of low expression of mRNA of IFNAR-1 and IFNAR-2. IFN-alpha was present in 14.2% (4/28) of the CIN samples but was not expressed in the control group. mRNA 2'5'OAS were expressed in 28.5% (8/28) of the CIN samples and 11.7% (2/17) of the control samples (not statistically significant). Fifty percent (14/28) of the CIN samples were positive for HPV DNA. Cervical biopsy samples from control women or those without neoplasia or HPV infection displayed higher IFN-alpha receptor expression than those with CIN, while simultaneous expression of both IFN-alpha receptor subunits was found only in the control group. There was no significant difference in mRNA expression of IFN-alpha and 2'5'OAS between the control and CIN groups. Then we concluded that the samples obtained from patients with CIN present low levels of the IFN-alpha receptor mRNA.

Construction of promoter-probe shuttle vectors for Escherichia coli and corynebacteria on the basis of promoterless alpha-amylase gene.

PubMed

Ugorcáková, J; Bukovská, G; Timko, J

2000-01-01

We constructed new promoter-probe vectors for E. coli and corynebacteria based on the promoterless alpha-amylase gene originating from Bacillus subtilis. Vectors pJUPAE1 and pJUPAE2 are suitable for isolation of transcriptionally active fragments from plasmids, phages or genomic DNA. alpha-Amylase activity can be easily visually detected on agar plates containing a chromogenic substrate, or by direct measurement of alpha-amylase activity.

Jellyfish mesogloea collagen. Characterization of molecules as alpha 1 alpha 2 alpha 3 heterotrimers.

PubMed

Miura, S; Kimura, S

1985-12-05

The mesogloea collagen of a primitive animal, the jellyfish Stomolophus nomurai, belonging to the class Scyphozoa in the Coelenterata, was studied with respect to its chain structure. Most of the mesogloea collagen was solubilized by limited digestion with pepsin and isolated by selective precipitation at 0.9 m NaCl in 0.5 M acetic acid. Upon denaturation, the pepsin-solubilized collagen produced three distinct alpha chains, alpha 1, alpha 2, and alpha 3, in comparable amounts which were separable by CM-cellulose chromatography. The nonidentity of these alpha chains was confirmed by amino acid and carbohydrate analyses and peptide mapping. Furthermore, the introduction of intramolecular cross-links into native molecules by formaldehyde yielded a large proportion of gamma 123 chain with chain structure alpha 1 alpha 2 alpha 3, as judged by chromatographic behavior and peptide maps. We concluded that mesogloea collagen is comprised of alpha 1 alpha 2 alpha 3 heterotrimers and is chemically like vertebrate Type V collagen. On the other hand, sea anemone mesogloea collagen from the class Anthozoa was previously reported to comprise (alpha)3 homotrimers (Katzman, R. L., and Kang, A. H. (1972) J. Biol. Chem. 247, 5486-5489). On the basis of these findings, we assume that alpha 1 alpha 2 alpha 3 heterotrimers arose in evolution with the divergence of Scyphozoa and Anthozoa.

MutY DNA Glycosylase Protects Cells From Tumor Necrosis Factor Alpha-Induced Necroptosis.

PubMed

Tran, An Hue Vy; Han, Se Hee; Kim, Joon; Grasso, Francesca; Kim, In San; Han, Ye Sun

2017-07-01

Numerous studies have implied that mutY DNA glycosylase (MYH) is involved in the repair of post-replicative mispairs and plays a critical role in the base excision repair pathway. Recent in vitro studies have shown that MYH interacts with tumor necrosis factor receptor type 1-associated death domain (TRADD), a key effector protein of tumor necrosis factor receptor-1 (TNFR1) signaling. The association between MYH and TRADD is reversed during tumor necrosis factor alpha (TNF-α)- and camptothecin (CPT)-induced apoptosis, and enhanced during TNF-α-induced survival. After investigating the role of MYH interacts with various proteins following TNF-α stimulation, here, we focus on MYH and TRADD interaction functions in necroptosis and its effects to related proteins. We report that the level of the MYH and TRADD complex was also reduced during necroptosis induced by TNF-α and zVAD-fmk. In particular, we also found that MYH is a biologically important necrosis suppressor. Under combined TNF-α and zVAD-fmk treatment, MYH-deficient cells were induced to enter the necroptosis pathway but primary mouse embryonic fibroblasts (MEFs) were not. Necroptosis in the absence of MYH proceeds via the inactivation of caspase-8, followed by an increase in the formation of the kinase receptor- interacting protein 1 (RIP1)-RIP3 complex. Our results suggested that MYH, which interacts with TRADD, inhibits TNF-α necroptotic signaling. Therefore, MYH inactivation is essential for necroptosis via the downregulation of caspase-8. J. Cell. Biochem. 118: 1827-1838, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Preliminary results on an H-alpha map of the Gum nebula obtained with the D-z-A satellite

NASA Technical Reports Server (NTRS)

Blamont, J. E.; Levasseur, A. C.

1971-01-01

Hydrogen emissions, including telluric H alpha, in the terrestrial atmosphere were studied. The H alpha experiment was designed to use a monochromatic photometer that would provide measurements of weak H alpha emission originating at altitudes of a few hundred or thousand km, free of contamination by other telluric emissions. The Gum nebula was recorded by the instrument as a much more intense feature than the geocoronal emission. The spacecraft employed and the optical onboard equipment are described. Field of view, spectral response, absolute calibration, linearity, and inflight performance are discussed.

Quantification of tamoxifen DNA adducts using on-line sample preparation and HPLC-electrospray ionization tandem mass spectrometry.

PubMed

Gamboa da Costa, Gonçalo; Marques, M Matilde; Beland, Frederick A; Freeman, James P; Churchwell, Mona I; Doerge, Daniel R

2003-03-01

The nonsteroidal antiestrogen tamoxifen is used as an adjuvant chemotherapeutic agent for the treatment of all stages of hormone-dependent breast cancer and more recently as a chemopreventive agent in women with elevated risk of developing the disease. While clearly beneficial for the treatment of breast cancer, tamoxifen has been reported to increase the risk of endometrial cancer in women. Furthermore, it has been shown to be hepatocarcinogenic in rats. Tamoxifen is clearly genotoxic in rat liver, as indicated by the formation of DNA adducts; the occurrence of tamoxifen DNA adducts in human endometrial tissue is more controversial. The detection and quantitation of tamoxifen DNA adducts have relied primarily upon (32)P-postlabeling, with other techniques, such as immunoassays and accelerator mass spectrometry, being used to a much lesser extent. To expand the range of available analytical methodologies for quantifying tamoxifen DNA adducts, we have developed an assay using on-line sample preparation, coupled with HPLC and electrospray ionization tandem mass spectrometry (ES-MS/MS). alpha-Acetoxytamoxifen was reacted with salmon testis DNA at ratios between 0.1 ng and 1 mg alpha-acetoxytamoxifen per mg DNA. After enzymatic hydrolysis to nucleosides, the most highly modified DNA samples were analyzed by HPLC-UV, which indicated the presence of two adduct peaks in approximately a 1:4 ratio. The major adduct was isolated, rigorously characterized as (E)-alpha-(deoxyguanosin-N(2)-yl)tamoxifen, and quantified on the basis of its molar extinction coefficient. A similar reaction was conducted with [N(CD(3))(2)]-alpha-acetoxytamoxifen to prepare a deuterated adduct that could serve as an internal standard for ES-MS/MS. The limit of detection for the HPLC-ES-MS/MS method was approximately 5 adducts/10(9) nucleotides, with an intra- and interassay precision of 3% relative standard deviation. The method was validated over the range of 8-1 520,000 adducts/10(8) nucleotides

Sequence-selective DNA cleavage by a chimeric metallopeptide.

PubMed

Kovacic, Roger T; Welch, Joel T; Franklin, Sonya J

2003-06-04

A chimeric metallopeptide derived from the sequences of two structurally superimposable motifs was designed as an artificial nuclease. Both DNA recognition and nuclease activity have been incorporated into a small peptide sequence. P3W, a 33-mer peptide comprising helices alpha2 and alpha3 from the engrailed homeodomain and the consensus EF-hand Ca-binding loop binds one equivalent of lanthanides or calcium and folds upon metal binding. The conditional formation constants (in the presence of 50 mM Tris) of P3W for Eu(III) (K(a) = (2.1 +/- 0.1) x 10(5) M(-1)) and Ce(IV) (K(a) = (2.6 +/- 0.1) x 10(5) M(-1)) are typical of isolated EF-hand peptides. Circular dichroism studies show that 1:1 CeP3W is 26% alpha-helical and EuP3W is up to 40% alpha-helical in the presence of excess metal. The predicted helicity of the folded peptide based on helix length and end effects is about 50%, showing the metallopeptides are significantly folded. EuP3W has considerably more secondary structure than our previously reported chimeras (Welch, J. T.; Sirish, M.; Lindstrom, K. M.; Franklin, S. J. Inorg. Chem. 2001, 40, 1982-1984). Eu(III)P3W and Ce(IV)P3W nick supercoiled DNA at pH 6.9, although EuP3W is more active at pH 8. CeP3W cleaves linearized, duplex DNA as well as supercoiled plasmid. The cleavage of a 5'-(32)P-labeled 121-mer DNA fragment was followed by polyacrylamide gel electrophoresis. The cleavage products are 3'-OPO(3) termini exclusively, suggesting a regioselective or multistep mechanism. In contrast, uncomplexed Ce(IV) and Eu(III) ions produce both 3'-OPO(3) and 3'-OH, and no evidence of 4'-oxidative cleavage termini with either metal. The complementary 3'-(32)P-labeled oligonucleotide experiment also showed both 5'-OPO(3) and 5'-OH termini were produced by the free ions, whereas CeP3W produces only 5'-OPO(3) termini. In addition to apparent regioselectivity, the metallopeptides cut DNA with modest sequence discrimination, which suggests that the HTH motif binds DNA as

Molecular study of electron transfer flavoprotein alpha-subunit deficiency in two Japanese children with different phenotypes of glutaric acidemia type II.

PubMed

Purevjav, E; Kimura, M; Takusa, Y; Ohura, T; Tsuchiya, M; Hara, N; Fukao, T; Yamaguchi, S

2002-09-01

Electron transfer flavoprotein is a mitochondrial matrix protein composed of alpha- and beta-subunits (ETF alpha and ETF beta, respectively). This protein transfers electrons between several mitochondrial dehydrogenases and the main respiratory chain via ETF dehydrogenase (ETF-DH). Defects in ETF or ETF-DH cause glutaric acidemias type II (GAII). We investigated the molecular basis of ETF alpha deficiency in two Japanese children with different clinical phenotypes using expression study. Patient 1 had the severe form of GAII, a compound heterozygote of two mutations: 799G to A (alpha G267R) and nonsense 7C to T (alpha R3X). Patient 2 had the mild form and carried two heterozygous mutations: 764G to T (alpha G255V) and 478delG (frameshift). Both patients had one each of missense mutations in one allele; the others were either nonsense or truncated. Restriction enzyme digestion assay using genomic DNAs from 100 healthy Japanese revealed that these mutations were all novel. No signal for ETF alpha was detected by immunoblotting in cases of missense mutants, while wild-type cDNA resulted in expression of ETF alpha protein. Transfection with wild-type ETF alpha cDNA into cultured cells from both patients elevated incorporation of radioisotope-labelled fatty acids. These four mutations were pathogenic for GAII and missense mutations, alpha G255V and alpha G267R were considered anecdotal for mild and severe forms, respectively.

Characterization of pancreatic lesions from MT-tgf alpha, Ela-myc and MT-tgf alpha/Ela-myc single and double transgenic mice.

PubMed

Liao, Dezhong Joshua; Wang, Yong; Wu, Jiusheng; Adsay, Nazmi Volkan; Grignon, David; Khanani, Fayyaz; Sarkar, Fazlul H

2006-07-05

In order to identify good animal models for investigating therapeutic and preventive strategies for pancreatic cancer, we analyzed pancreatic lesions from several transgenic models and made a series of novel findings. Female MT-tgf alpha mice of the MT100 line developed pancreatic proliferation, acinar-ductal metaplasia, multilocular cystic neoplasms, ductal adenocarcinomas and prominent fibrosis, while the lesions in males were less severe. MT-tgf alpha-ES transgenic lines of both sexes developed slowly progressing lesions that were similar to what was seen in MT100 males. In both MT100 and MT-tgf alpha-ES lines, TGF alpha transgene was expressed mainly in proliferating ductal cells. Ela-myc transgenic mice with a mixed C57BL/6, SJL and FVB genetic background developed pancreatic tumors at 2-7 months of age, and half of the tumors were ductal adenocarcinomas, similar to what was reported originally by Sandgren et al 1. However, in 20% of the mice, the tumors metastasized to the liver. MT100/Ela-myc and MT-tgf alpha-ES/Ela-myc double transgenic mice developed not only acinar carcinomas and mixed carcinomas as previously reported but also various ductal-originated lesions, including multilocular cystic neoplasms and ductal adenocarcinomas. The double transgenic tumors were more malignant and metastasized to the liver at a higher frequency (33%) compared with the Ela-myc tumors. Sequencing of the coding region of p16ink4, k-ras and Rb cDNA in small numbers of pancreatic tumors did not identify mutations. The short latency for tumor development, the variety of tumor morphology and the liver metastases seen in Ela-myc and MT-tgf alpha/Ela-myc mice make these animals good models for investigating new therapeutic and preventive strategies for pancreatic cancer.

Mycophenolic acid attenuates tumor necrosis factor-alpha-induced endothelin-1 production in human aortic endothelial cells.

PubMed

Yang, Won Seok; Lee, Joo Mi; Han, Nam Jeong; Kim, Yoon Ji; Chang, Jai Won; Park, Su-Kil

2010-07-01

Atherosclerotic cardiovascular disease is the major cause of morbidity and mortality in solid organ transplant recipients. Endothelin-1 (ET-1) is implicated in the pathogenesis of atherosclerosis and is one of the potential therapeutic targets. This study was conducted to evaluate the effect of mycophenolic acid (MPA), an immunosuppressant for the transplant recipients, on tumor necrosis factor-alpha (TNF-alpha)-induced ET-1 production in aortic endothelial cells. In cultured human aortic endothelial cells, TNF-alpha increased ET-1 through AP-1 and NF-kappaB, whereas MPA attenuated it by reducing both AP-1 and NF-kappaB DNA-binding activities. TNF-alpha increased ET-1 via c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), but not extracellular signal-regulated kinase. N-acetylcysteine that downregulated TNF-alpha-induced reactive oxygen species (ROS) inhibited JNK activation, but not p38 MAPK. N-acetylcysteine, SP600125 (JNK inhibitor) and SB203580 (p38 MAPK inhibitor) attenuated TNF-alpha-induced DNA-binding activities of both AP-1 and NF-kappaB. MPA inhibited JNK and p38 MAPK activations as well as ROS generation. N-acetylcysteine, SP600125, SB203580 and MPA had no effect on either TNF-alpha-induced IkappaBalpha degradation or p65 nuclear translocation, but attenuated p65 Ser276 phosphorylation. MPA attenuated TNF-alpha-induced ET-1 production through inhibitions of ROS-dependent JNK and ROS-independent p38 MAPK that regulated NF-kappaB as well as AP-1. These findings suggest that MPA could have an effect of amelioration of atherosclerosis. Copyright (c) 2010 Elsevier Ireland Ltd. All rights reserved.

Human Gamma Satellite Insulator Sequences to Prevent Gene Silencing | NCI Technology Transfer Center | TTC

Cancer.gov

This invention describes the use of chromatin insulators, or gamma satellite DNA, to inhibit gene silencing in a cell, which may have a significant impact on gene therapy across multiple diseases where gene silencing is the cause. Experimental data has demonstrated these gamma satellite DNAs overcome gene position effects and ultimately inhibit gene silencing.

Progesterone's 5 alpha-reduced metabolite, 3 alpha,5 alpha-THP, mediates lateral displacement of hamsters.

PubMed

Frye, Cheryl A; Rhodes, Madeline E

2005-03-15

5 alpha-Pregnan-3 alpha-ol-20-one (3 alpha,5 alpha-THP), progesterone (P4)'s 5 alpha-reduced, 3 alpha-hydroxysteroid oxidoreduced product, facilitates lordosis of rodents in part via agonist-like actions at GABA(A)/benzodiazepine receptor complexes in the ventral tegmental area (VTA). Whether 3 alpha,5 alpha-THP influences another reproductively-relevant behavior, lateral displacement, of hamsters was investigated. Lateral displacement is the movement that female hamsters make with their perineum towards male-like tactile stimulation. This behavior facilitates, and is essential for, successful mating. Hamsters in behavioral estrus had greater lateral displacement responses when endogenous progestin levels were elevated compared to when progestin levels were lower. Administration of P4, a prohormone for 3 alpha,5 alpha-THP, dose-dependently (500 > 200 > 100, 50, or 0 microg) enhanced lateral displacement of ovariectomized hamsters that had been primed with SC estradiol benzoate (5 or 10 microg). Inhibiting P4's metabolism to 3 alpha,5 alpha-THP by co-administering finasteride, a 5 alpha-reductase inhibitor, or indomethacin, a 3 alpha-hydroxysteroid oxidoreductase inhibitor, either systemically or to the VTA, significantly decreased lateral displacement and midbrain progestin levels of naturally receptive or hormone-primed hamsters compared to controls. These data suggest that lateral displacement is progestin-sensitive and requires the formation of 3 alpha,5 alpha-THP in the midbrain VTA.

A SERS-active sensor based on heterogeneous gold nanostar core-silver nanoparticle satellite assemblies for ultrasensitive detection of aflatoxinB1

NASA Astrophysics Data System (ADS)

Li, Aike; Tang, Lijuan; Song, Dan; Song, Shanshan; Ma, Wei; Xu, Liguang; Kuang, Hua; Wu, Xiaoling; Liu, Liqiang; Chen, Xin; Xu, Chuanlai

2016-01-01

A surface-enhanced Raman scattering (SERS) sensor based on gold nanostar (Au NS) core-silver nanoparticle (Ag NP) satellites was fabricated for the first time to detect aflatoxinB1 (AFB1). We constructed the SERS sensor using AFB1 aptamer (DNA1)-modified Ag satellites and a complementary sequence (DNA2)-modified Au NS core. The Raman label (ATP) was modified on the surface of Ag satellites. The SERS signal was enhanced when the satellite NP was attached to the Au core NS. The AFB1 aptamer on the surface of Ag satellites would bind to the targets when AFB1 was present in the system, Ag satellites were then removed and the SERS signal decreased. This SERS sensor showed superior specificity for AFB1 and the linear detection range was from 1 to 1000 pg mL-1 with the limit of detection (LOD) of 0.48 pg mL-1. The excellent recovery experiment using peanut milk demonstrated that the sensor could be applied in food and environmental detection.A surface-enhanced Raman scattering (SERS) sensor based on gold nanostar (Au NS) core-silver nanoparticle (Ag NP) satellites was fabricated for the first time to detect aflatoxinB1 (AFB1). We constructed the SERS sensor using AFB1 aptamer (DNA1)-modified Ag satellites and a complementary sequence (DNA2)-modified Au NS core. The Raman label (ATP) was modified on the surface of Ag satellites. The SERS signal was enhanced when the satellite NP was attached to the Au core NS. The AFB1 aptamer on the surface of Ag satellites would bind to the targets when AFB1 was present in the system, Ag satellites were then removed and the SERS signal decreased. This SERS sensor showed superior specificity for AFB1 and the linear detection range was from 1 to 1000 pg mL-1 with the limit of detection (LOD) of 0.48 pg mL-1. The excellent recovery experiment using peanut milk demonstrated that the sensor could be applied in food and environmental detection. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr08372a

Cloning and characterization of the rat HIF-1 alpha prolyl-4-hydroxylase-1 gene.

PubMed

Cobb, Ronald R; McClary, John; Manzana, Warren; Finster, Silke; Larsen, Brent; Blasko, Eric; Pearson, Jennifer; Biancalana, Sara; Kauser, Katalin; Bringmann, Peter; Light, David R; Schirm, Sabine

2005-08-01

Prolyl-4-hydroxylase domain-containing enzymes (PHDs) mediate the oxygen-dependent regulation of the heterodimeric transcription factor hypoxia-inducible factor-1 (HIF-1). Under normoxic conditions, one of the subunits of HIF-1, HIF-1alpha, is hydroxylated on specific proline residues to target HIF-1alpha for degradation by the ubiquitin-proteasome pathway. Under hypoxic conditions, the hydroxylation by the PHDs is attenuated by lack of the oxygen substrate, allowing HIF-1 to accumulate, translocate to the nucleus, and mediate HIF-mediated gene transcription. In several mammalian species including humans, three PHDs have been identified. We report here the cloning of a full-length rat cDNA that is highly homologous to the human and murine PHD-1 enzymes and encodes a protein that is 416 amino acids long. Both cDNA and protein are widely expressed in rat tissues and cell types. We demonstrate that purified and crude baculovirus-expressed rat PHD-1 exhibits HIF-1alpha specific prolyl hydroxylase activity with similar substrate affinities and is comparable to human PHD-1 protein.

Pur-alpha regulates cytoplasmic stress granule dynamics and ameliorates FUS toxicity

PubMed Central

Daigle, J Gavin; Krishnamurthy, Karthik; Ramesh, Nandini; Casci, Ian; Monaghan, John; McAvoy, Kevin; Godfrey, Earl W; Daniel, Dianne C.; Johnson, Edward M.; Monahan, Zach; Shewmaker, Frank; Pasinelli, Piera; Pandey, Udai Bhan

2016-01-01

Amyotrophic lateral Sclerosis is characterized by progressive loss of motor neurons in the brain and spinal cord. Mutations in several genes, including FUS, TDP43, Matrin 3, hnRNPA2 and other RNA binding proteins, have been linked to ALS pathology. Recently, Pur-alpha a DNA/RNA binding protein was found to bind to C9orf72 repeat expansions and could possibly play a role in the pathogenesis of ALS. When overexpressed, Pur-alpha mitigates toxicities associated with Fragile X tumor ataxia syndrome (FXTAS) and C9orf72 repeat expansion diseases in Drosophila and mammalian cell culture models. However, the function of Pur-alpha in regulating ALS pathogenesis has not been fully understood. We identified Pur-alpha as a novel component of cytoplasmic stress granules (SGs) in ALS patient cells carrying disease-causing mutations in FUS. When cells were challenged with stress, we observed that Pur-alpha co-localized with mutant FUS in ALS patient cells and became trapped in constitutive SGs. We also found that FUS physically interacted with Pur-alpha in mammalian neuronal cells. Interestingly, shRNA mediated knock down of endogenous Pur-alpha significantly reduced formation of cytoplasmic stress granules in mammalian cells suggesting that Pur-alpha is essential for the formation of SGs. Furthermore, ectopic expression of Pur-alpha blocked cytoplasmic mislocalization of mutant FUS and strongly suppressed toxicity associated with mutant FUS expression in primary motor neurons. Our data emphasizes the importance of stress granules in ALS pathogenesis and identifies Pur-alpha as a novel regulator of SG dynamics. PMID:26728149

Distinct modes of regulation of transcription of hepatitis B virus by the nuclear receptors HNF4alpha and COUP-TF1.

PubMed

Yu, Xianming; Mertz, Janet E

2003-02-01

To study the effects of the nuclear receptors (NRs) HNF4alpha and COUP-TF1 on the life cycle of hepatitis B virus (HBV), the human hepatoma cell line Huh7 was transiently cotransfected with plasmids containing the HBV genome and encoding these two NRs. Overexpression of HNF4alpha and COUP-TF1 led to a 9-fold increase and a 7- to 10-fold decrease, respectively, in viral DNA synthesis. These two NRs also exhibited distinct modes of regulation of viral transcription. Overexpression of HNF4alpha led to a more-than-10-fold increase in synthesis of the pregenomic RNA but to only a 2- to 3-fold increase in synthesis of the pre-C and S RNAs. Moreover, the NR response element within the pre-C promoter, NRRE(preC,) played the major role in activation of pregenomic RNA synthesis by HNF4alpha. On the other hand, overexpression of COUP-TF1 led to an over-10-fold repression of synthesis of both pre-C and pregenomic RNAs mediated through either NRRE(preC) or NRRE(enhI). HNF4alpha and COUP-TF1 antagonized each other's effects on synthesis of pregenomic RNA and viral DNA when they were co-overexpressed. A naturally occurring HBV variant which allows for binding by HNF4alpha but not COUP-TF1 in its NRRE(preC) exhibited significantly higher levels of synthesis of pregenomic RNA and viral DNA than wild-type HBV in coexpression experiments. Last, deletion analysis revealed that non-NRRE sequences located within both the C and pre-S1 regions are also essential for maximum activation of the pregenomic promoter by HNF4alpha but not for repression by COUP-TF1. Thus, HNF4alpha and COUP-TF1 function through different mechanisms to regulate expression of the HBV genes.

Synthesis of methyl 3-O-alpha-D-mannopyranosyl-alpha-D-talopyranoside and methyl 3-O-alpha-D-talopyranosyl-alpha-D-talopyranoside.

PubMed

Dubey, R; Jain, R K; Abbas, S A; Matta, K L

1987-08-01

Methyl 2-O-benzyl-3-O-(2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl)-alpha- D-mannopyranoside (4) and methyl 2-O-benzyl-3-O-alpha-D-mannopyranosyl-alpha-D-mannopyranoside (6) were prepared from a common intermediate, namely, methyl 2-O-benzyl-4,6-O-benzylidene-3-O-(2,3,4,6-tetra-O-acetyl-alpha-D- mannopyranosyl)-alpha-D-mannopyranoside. On treatment with tert-butylchlorodiphenylsilane, in N,N-dimethylformamide in the presence of imidazole, 4 and 6 afforded methyl 2-O-benzyl-6-O-tert-butyldiphenylsilyl-3-O-(2,3,4,6-tetra-O-acetyl -alpha-D- mannopyranosyl)-alpha-D-mannopyranoside (7), and methyl 2-O-benzyl-6-O-tert-butyldiphenylsilyl-3-O-(6-O-tert- butyldiphenylsilyl-alpha-D-mannopyranosyl)-alpha-D-mannopyranoside (8), respectively. Compound 8 was converted into its 2,3-O-isopropylidene derivative (9), and oxidation of 7 and 9 with pyridinium chlorochromate, and reduction of the resulting carbonyl intermediates gave methyl 2-O-benzyl-6-O-tert-butyldiphenylsilyl-3-O-(2,3,4,6-tetra-O-acetyl -alpha-D- mannopyranosyl)-alpha-D-talopyranoside and methyl 2-O-benzyl-6-O-tert-butyldiphenylsilyl-3-O-(6-O-tert-butyldiphe nylsilyl- 2,3-O-isopropylidene-alpha-D-talopyranosyl)-alpha-D-talopyranoside , respectively. Removal of the protecting groups furnished the title disaccharides.

Expression and kinetic properties of a recombinant 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase isoenzyme of human liver.

PubMed

Deyashiki, Y; Tamada, Y; Miyabe, Y; Nakanishi, M; Matsuura, K; Hara, A

1995-08-01

Human liver cytosol contains multiple forms of 3 alpha-hydroxysteroid dehydrogenase and dihydrodiol dehydrogenase with hydroxysteroid dehydrogenase activity, and multiple cDNAs for the enzymes have been cloned from human liver cDNA libraries. To understand the relationship of the multiple enzyme froms to the genes, a cDNA, which has been reported to code for an isoenzyme of human liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase, was expressed in Escherichia coli. The recombinant enzyme showed structural and functional properties almost identical to those of the isoenzyme purified from human liver. In addition, the recombinant isoenzyme efficiently reduced 5 alpha-dihydrotestosterone and 5 beta-dihydrocortisone, the known substrates of human liver 3 alpha-hydroxysteroid dehydrogenase and chlordecone reductase previously purified, which suggests that these human liver enzymes are identical. Furthermore, the steady-state kinetic data for NADP(+)-linked (S)-1-indanol oxidation by the recombinant isoenzyme were consistent with a sequential ordered mechanism in which NADP+ binds first. Phenolphthalein inhibited this isoenzyme much more potently than it did the other human liver dihydrodiol dehydrogenases, and was a competitive inhibitor (Ki = 20 nM) that bound to the enzyme-NADP+ complex.

Genomic in situ hybridization in interspecific hybrids of scallops (Bivalvia, Pectinidae) and localization of the satellite DNA Cf303, and the vertebrate telomeric sequences (TTAGGG)n on chromosomes of scallop Chlamys farreri (Jones & Preston, 1904)

PubMed Central

Hu, Liping; Jiang, Liming; Bi, Ke; Liao, Huan; Yang, Zujing; Huang, Xiaoting; Bao, Zhenmin

2018-01-01

Abstract Mitotic chromosome preparations of the interspecific hybrids Chlamys farreri (Jones & Preston, 1904) × Patinopecten yessoensis (Jay, 1857), C. farreri × Argopecten irradians (Lamarck, 1819) and C. farreri × Mimachlamys nobilis (Reeve, 1852) were used to compare two different scallop genomes in a single slide. Although genomic in situ hybridization (GISH) using genomic DNA from each scallop species as probe painted mitotic chromosomes of the interspecific hybrids, the painting results were not uniform; instead it showed species-specific distribution patterns of fluorescent signals among the chromosomes. The most prominent GISH-bands were mainly located at centromeric or telomeric regions of scallop chromosomes. In order to illustrate the sequence constitution of the GISH-bands, the satellite Cf303 sequences of C. farreri and the vertebrate telomeric (TTAGGG)n sequences were used to map mitotic chromosomes of C. farreri by fluorescence in situ hybridization (FISH). The results indicated that the GISH-banding pattern presented by the chromosomes of C. farreri is mainly due to the distribution of the satellite Cf303 DNA, therefore suggesting that the GISH-banding patterns found in the other three scallops could also be the result of the chromosomal distribution of other species-specific satellite DNAs. PMID:29675138

Ginsenoside Rf, a component of ginseng, regulates lipoprotein metabolism through peroxisome proliferator-activated receptor {alpha}

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Hyunghee; Gonzalez, Frank J.; Yoon, Michung

We investigated whether ginseng regulates lipoprotein metabolism by altering peroxisome proliferator-activated receptor {alpha} (PPAR{alpha})-mediated pathways, using a PPAR{alpha}-null mouse model. Administration of ginseng extract, ginsenosides, and ginsenoside Rf (Rf) to wild-type mice not only significantly increased basal levels of hepatic apolipoprotein (apo) A-I and C-III mRNA compared with wild-type controls, but also substantially reversed the reductions in mRNA levels of apo A-I and C-III expected following treatment with the potent PPAR{alpha} ligand Wy14,643. In contrast, no effect was detected in the PPAR{alpha}-null mice. Testing of eight main ginsenosides on PPAR{alpha} reporter gene expression indicated that Rf was responsible for themore » effects of ginseng on lipoprotein metabolism. Furthermore, the inhibition of PPAR{alpha}-dependent transactivation by Rf seems to occur at the level of DNA binding. These results demonstrate that ginseng component Rf regulates apo A-I and C-III mRNA and the actions of Rf on lipoprotein metabolism are mediated via interactions with PPAR{alpha}.« less

Sarcolemmal alpha and gamma sarcoglycan protein deficiencies in Turkish siblings with a novel missense mutation in the alpha sarcoglycan gene.

PubMed

Diniz, Gulden; Tosun Yildirim, Hulya; Akinci, Gulcin; Hazan, Filiz; Ozturk, Aysel; Yararbas, Kanay; Tukun, Ajlan

2014-06-01

The sarcoglycan alpha gene, also known as the adhalin gene, is located on chromosome 17q21; mutations in this gene are associated with limb-girdle muscular dystrophy type 2D. We describe two Turkish siblings with findings consistent with limb-girdle muscular dystrophy type 2D. The evaluation excluded a dystrophinopathy, which is the most common form of muscular dystrophy. Both siblings had very high levels of creatinine phosphokinase and negative molecular tests for deletions and duplications of the dystrophin gene. The older boy presented at 8 years of age with an inability to climb steps and an abnormal gait. His younger brother was 5 years old and had similar symptoms. The muscle biopsy evaluation was performed only in the older brother. The muscle biopsy showed dystrophic features as well as a deficiency in the expression of two different glycoproteins: the alpha sarcoglycan and the gamma sarcoglycan. Sarcolemmal expressions of dystrophin and other sarcoglycans (beta and delta) were diffusely present. DNA analysis demonstrated the presence of previously unknown homozygous mutations [c.226 C > T (p.L76 F)] in exon 3 in the sarcoglycan alpha genes of both siblings. Similar heterozygous point mutations at the same locus were found in both parents, but the genes of beta, delta, and gamma sarcoglycan were normal in the remaining family members. We describe two siblings with limb-girdle muscular dystrophy type 2D with a novel missense mutation. These patients illustrate that the differential diagnosis of muscular dystrophies is impossible with clinical findings alone. Therefore, a muscle biopsy and DNA analysis remain essential methods for diagnosis of muscle diseases. Copyright © 2014 Elsevier Inc. All rights reserved.

Nucleotide sequences of bovine alpha S1- and kappa-casein cDNAs.

PubMed Central

Stewart, A F; Willis, I M; Mackinlay, A G

1984-01-01

The nucleotide sequences corresponding to bovine alpha S1- and kappa-casein mRNAs are presented. An unusual alpha S1-casein cDNA has been characterised whose 5' end commences upstream from its putative TATA box. The alpha S1-casein mRNA is compared to rat alpha-casein mRNA and two components of divergence are identified. Firstly, the two sequences have diverged at a high point mutation rate and the rate of amino acid replacement by this mechanism is at least as great as the rate of divergence of any other part of the mRNAs. Secondly, the protein coding sequence has been subjected to several insertion/deletion events, one of which may be an example of exon shuffling . The kappa-casein mRNA sequence verifies the proposition that it has arisen from a different ancestral gene to the other caseins. Images PMID:6328443

An alternative method for cDNA cloning from surrogate eukaryotic cells transfected with the corresponding genomic DNA.

PubMed

Hu, Lin-Yong; Cui, Chen-Chen; Song, Yu-Jie; Wang, Xiang-Guo; Jin, Ya-Ping; Wang, Ai-Hua; Zhang, Yong

2012-07-01

cDNA is widely used in gene function elucidation and/or transgenics research but often suitable tissues or cells from which to isolate mRNA for reverse transcription are unavailable. Here, an alternative method for cDNA cloning is described and tested by cloning the cDNA of human LALBA (human alpha-lactalbumin) from genomic DNA. First, genomic DNA containing all of the coding exons was cloned from human peripheral blood and inserted into a eukaryotic expression vector. Next, by delivering the plasmids into either 293T or fibroblast cells, surrogate cells were constructed. Finally, the total RNA was extracted from the surrogate cells and cDNA was obtained by RT-PCR. The human LALBA cDNA that was obtained was compared with the corresponding mRNA published in GenBank. The comparison showed that the two sequences were identical. The novel method for cDNA cloning from surrogate eukaryotic cells described here uses well-established techniques that are feasible and simple to use. We anticipate that this alternative method will have widespread applications.

Synthesis of methyl 2-O-alpha-D-mannopyranosyl-alpha-D-talopyranoside and methyl 2-O-alpha-D-talopyranosyl-alpha-D-talopyranoside.

PubMed

Jain, R K; Dubey, R; Abbas, S A; Matta, K L

1987-03-15

Treatment of methyl 3-O-benzyl-2-O-(2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl)-alpha-D- mannopyranoside (1) with tert-butyldiphenylsilyl chloride in N,N-dimethylformamide afforded methyl 3-O-benzyl-6-O-tert-butyldiphenylsilyl-2-O-(2,3,4,6-tetra-O-acetyl -alpha-D- mannopyranosyl)-alpha-D-mannopyranoside (2). Oxidation of 2 with pyridinium chlorochromate, followed by reduction of the carbonyl group, and subsequent O-deacetylation afforded methyl 3-O-benzyl-6-O-tert-butyldiphenylsilyl-2-O-alpha-D-mannopyranosyl- alpha-D- talopyranoside (5). Cleavage of the tert-butyldiphenylsilyl group of 5 with tetrabutylammonium fluoride in oxolane, followed by hydrogenolysis, gave methyl 2-O-alpha-D-mannopyranosyl-alpha-D-talopyranoside (7). O-Deacetylation of 1 gave methyl 3-O-benzyl-2-O-alpha-D-mannopyranosyl-alpha-D-mannopyranoside (8). Treatment of 8 with tert-butyldiphenylsilyl chloride afforded a 6,6'-disilyl derivative, which was converted into a 2',3'-O-isopropylidene derivative, and then further oxidized with pyridinium chlorochromate. The resulting diketone was reduced and removal of the protecting groups gave methyl 2-O-alpha-D-talopyranosyl-alpha-D-talopyranoside (15). The structures of both 7 and 15 were established by 13C-n.m.r. spectroscopy.

Molecular cloning and characterization of satellite DNA sequences from constitutive heterochromatin of the habu snake (Protobothrops flavoviridis, Viperidae) and the Burmese python (Python bivittatus, Pythonidae).

PubMed

Matsubara, Kazumi; Uno, Yoshinobu; Srikulnath, Kornsorn; Seki, Risako; Nishida, Chizuko; Matsuda, Yoichi

2015-12-01

Highly repetitive DNA sequences of the centromeric heterochromatin provide valuable molecular cytogenetic markers for the investigation of genomic compartmentalization in the macrochromosomes and microchromosomes of sauropsids. Here, the relationship between centromeric heterochromatin and karyotype evolution was examined using cloned repetitive DNA sequences from two snake species, the habu snake (Protobothrops flavoviridis, Crotalinae, Viperidae) and Burmese python (Python bivittatus, Pythonidae). Three satellite DNA (stDNA) families were isolated from the heterochromatin of these snakes: 168-bp PFL-MspI from P. flavoviridis and 196-bp PBI-DdeI and 174-bp PBI-MspI from P. bivittatus. The PFL-MspI and PBI-DdeI sequences were localized to the centromeric regions of most chromosomes in the respective species, suggesting that the two sequences were the major components of the centromeric heterochromatin in these organisms. The PBI-MspI sequence was localized to the pericentromeric region of four chromosome pairs. The PFL-MspI and the PBI-DdeI sequences were conserved only in the genome of closely related species, Gloydius blomhoffii (Crotalinae) and Python molurus, respectively, although their locations on the chromosomes were slightly different. In contrast, the PBI-MspI sequence was also in the genomes of P. molurus and Boa constrictor (Boidae), and additionally localized to the centromeric regions of eight chromosome pairs in B. constrictor, suggesting that this sequence originated in the genome of a common ancestor of Pythonidae and Boidae, approximately 86 million years ago. The three stDNA sequences showed no genomic compartmentalization between the macrochromosomes and microchromosomes, suggesting that homogenization of the centromeric and/or pericentromeric stDNA sequences occurred in the macrochromosomes and microchromosomes of these snakes.

Genomic organization of the rat alpha 2u-globulin gene cluster.

PubMed

McFadyen, D A; Addison, W; Locke, J

1999-05-01

The alpha 2u-globulin are a group of similar proteins, belonging to the lipocalin superfamily of proteins, that are synthesized in a subset of secretory tissues in rats. The many alpha 2u-globulin isoforms are encoded by a multigene family that exhibits extensive homology. Despite a high degree of sequence identity, individual family members show diverse expression patterns involving complex hormonal, tissue-specific, and developmental regulation. Analysis suggests that there are approximately 20 alpha 2u-globulin genes in the rat genome. We have used fluorescence in situ hybridization (FISH) to show that the alpha 2u-globulin genes are clustered at a single site on rat Chromosome (Chr) 5 (5q22-24). Southern blots of rat genomic DNA separated by pulsed field gel electrophoresis indicated that the alpha 2u-globulin genes are contained on two NruI fragments with a total size of 880 kbp. Analysis of three P1 clones containing alpha 2u-globulin genes indicated that the alpha 2u-globulin genes are tandemly arranged in a head-to-tail fashion. The organization of the alpha 2u-globulin genes in the rat as a tandem array of single genes differs from the homologous major urinary protein genes in the mouse, which are organized as tandem arrays of divergently oriented gene pairs. The structure of these gene clusters may have consequences for the proposed function, as a pheromone transporter, for the protein products encoded by these genes.

Synthesis of 3 alpha, 5 alpha-tetrahydroaldosterone.

PubMed

Harnik, M; Kashman, Y; Morris, D J

1984-06-01

3 alpha, 5 alpha-Tetrahydroaldosterone (12a), a metabolite of aldosterone, has been synthesized from the lactone 2a or, preferably 11 beta, 21-dihydroxy-5-pregnene-3,20-dione-18-oic acid 3,20-di-(ethylene glycol)-ketal (18----11) lactone 21-acetate (6b), via 11 beta, 21-dihydroxy-5 alpha-pregnane-3,20-dione-18-oic acid 3,20-di-(ethylene glycol) ketal (18----11) lactone 21-acetate (4b). Selective hydrolysis of the latter at C-3 furnished the monoketal 5 which, on reduction with potassium tri-sec-butylborohydride, yielded predominantly 3 alpha, 11 beta, 21-trihydroxy-5 alpha-pregnan-20-one-18-oic acid 20-(ethylene glycol)-ketal (18----11) lactone (8a) and its acetate 8b. Further reduction with diisobutylaluminum hydride afforded 3 alpha, 5 alpha-tetrahydroaldosterone-20-ketal (10a), which was directly hydrolyzed to 12a with dilute acid in tetrahydrofuran-dioxan. Periodate oxidation led to the gamma-etiolactone 14a, which was then further converted into 5 alpha-dihydroaldosterone-gamma-etiolactone (14c).

Effects of trichostatin A on In vitro development and DNA methylation level of the satellite I region of swamp buffalo (Bubalus bubalis) cloned embryos.

PubMed

Srirattana, Kanokwan; Ketudat-Cairns, Mariena; Nagai, Takashi; Kaneda, Masahiro; Parnpai, Rangsun

2014-01-01

Trichostatin A (TSA), a histone deacetylase inhibitor, has been widely used to improve the cloning efficiency in several species. This brings our attention to investigation of the effects of TSA on developmental potential of swamp buffalo cloned embryos. Swamp buffalo cloned embryos were produced by electrical pulse fusion of male swamp buffalo fibroblasts with swamp buffalo enucleated oocytes. After fusion, reconstructed oocytes were treated with 0, 25 or 50 nM TSA for 10 h. The results showed that there was no significant difference in the rates of fusion (82-85%), cleavage (79-84%) and development to the 8-cell stage (59-65%) among treatment groups. The highest developmental rates to the morula and blastocyst stages of embryos were found in the 25 nM TSA-treated group (42.7 and 30.1%, respectively). We also analyzed the DNA methylation level in the satellite I region of donor cells and in in vitro fertilized (IVF) and cloned embryos using the bisulfite DNA sequencing method. The results indicated that the DNA methylation levels in cloned embryos were significantly higher than those of IVF embryos but approximately similar to those of donor cells. Moreover, there was no significant difference in the methylation level among TSA-treated and untreated cloned embryos. Thus, TSA treatments at 25 nM for 10 h could enhance the in vitro developmental potential of swamp buffalo cloned embryos, but no beneficial effect on the DNA methylation level was observed.

Effects of Trichostatin A on In Vitro Development and DNA Methylation Level of the Satellite I Region of Swamp Buffalo (Bubalus bubalis) Cloned Embryos

PubMed Central

SRIRATTANA, Kanokwan; KETUDAT-CAIRNS, Mariena; NAGAI, Takashi; KANEDA, Masahiro; PARNPAI, Rangsun

2014-01-01

Trichostatin A (TSA), a histone deacetylase inhibitor, has been widely used to improve the cloning efficiency in several species. This brings our attention to investigation of the effects of TSA on developmental potential of swamp buffalo cloned embryos. Swamp buffalo cloned embryos were produced by electrical pulse fusion of male swamp buffalo fibroblasts with swamp buffalo enucleated oocytes. After fusion, reconstructed oocytes were treated with 0, 25 or 50 nM TSA for 10 h. The results showed that there was no significant difference in the rates of fusion (82–85%), cleavage (79–84%) and development to the 8-cell stage (59–65%) among treatment groups. The highest developmental rates to the morula and blastocyst stages of embryos were found in the 25 nM TSA-treated group (42.7 and 30.1%, respectively). We also analyzed the DNA methylation level in the satellite I region of donor cells and in in vitro fertilized (IVF) and cloned embryos using the bisulfite DNA sequencing method. The results indicated that the DNA methylation levels in cloned embryos were significantly higher than those of IVF embryos but approximately similar to those of donor cells. Moreover, there was no significant difference in the methylation level among TSA-treated and untreated cloned embryos. Thus, TSA treatments at 25 nM for 10 h could enhance the in vitro developmental potential of swamp buffalo cloned embryos, but no beneficial effect on the DNA methylation level was observed. PMID:24909601

Efficient repair of DNA double-strand breaks in malignant cells with structural instability.

PubMed

Cheng, Yue; Zhang, Zhenhua; Keenan, Bridget; Roschke, Anna V; Nakahara, Kenneth; Aplan, Peter D

2010-01-05

Aberrant repair of DNA double-strand breaks (DSBs) is thought to be important in the generation of gross chromosomal rearrangements (GCRs). To examine how DNA DSBs might lead to GCRs, we investigated the repair of a single DNA DSB in a structurally unstable cell line. An I-SceI recognition site was introduced into OVCAR-8 cells between a constitutive promoter (EF1alpha) and the Herpes simplex virus thymidine kinase (TK) gene, which confers sensitivity to gancyclovir (GCV). Expression of I-SceI in these cells caused a single DSB. Clones with aberrant repair could acquire resistance to GCV by separation of the EF1alpha promoter from the TK gene, or deletion of either the EF1alpha promoter or the TK gene. All mutations that we identified were interstitial deletions. Treatment of cells with etoposide or bleomycin, agents known to produce DNA DSBs following expression of I-SceI also did not generate GCRs. Because we identified solely interstitial deletions using the aforementioned negative selection system, we developed a positive selection system to produce GCR. A construct containing an I-SceI restriction site immediately followed by a hygromycin phosphotransferase cDNA, with no promoter, was stably integrated into OVCAR-8 cells. DNA DSBs were produced by an I-SceI expression vector. None of the hygromycin resistant clones recovered had linked the hygromycin phosphotransferase cDNA to an endogenous promoter, but had instead captured a portion of the I-SceI expression vector. These results indicate that even in a structurally unstable malignant cell line, the majority of DNA DSBs are repaired by religation of the two broken chromosome ends, without the introduction of a GCR.

MODULATION BY IONIC STRENGTH AND SUPERHELICITY OF BENZO[a]PYRENE DIOL EPOXIDE INDUCED DNA ALKYLATION AND UNWINDING

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gamper, Howard B.; Straub, Kenneth; Calvin, Melvin

Superhelical and partially relaxed SV40 DNA were reacted in vitro with (+)7{beta}, 8{alpha}-dihydroxy-9{alpha},10{alpha}-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BaP diol epoxide). The modified DNA contained N{sup 2} guanine and N{sup 6} adeninte hydrocarbon adducts in the ratio 86:14. Superhelical SV40 DNA was approximately 6% more susceptible to modification than partially relaxed viral DNA. Counterions inhibited DNA alkylation by up to 90%, Mg{sup 2+} being 50-fold more effective than Na{sup +}. The sensitivity of covalent binding to helix stability is consistent with a reaction complex in which BaP diol epoxide is intercalated. The superhelical density of the modified DNA substrates was determined electrophoretically relative to partiallymore » relaxed standards and an unwinding angle for the hydrocarbon adducts was calculated. The angle was dependent upon the superhelicity of the DNA molecule and ranged from 330{sup o} to 30{sup o}. This data indicates that the modified base pairs are disrupted and, in the presence of torsional strain, act as centers for the further denaturation of up to 8 adjacent base pairs. In the absence of such strain the alkylation sites have an ordered structure with the attached hydrocarbon probably oriented in the minor or major groove of the helix.« less

Mutants in the mouse NuRD/Mi2 component P66alpha are embryonic lethal.

PubMed

Marino, Susan; Nusse, Roel

2007-06-13

The NuRD/Mi2 chromatin complex is involved in histone modifications and contains a large number of subunits, including the p66 protein. There are two mouse and human p66 paralogs, p66alpha and p66beta. The functions of these genes are not clear, in part because there are no mutants available, except in invertebrate model systems. We made loss of function mutants in the mouse p66alpha gene (mp66alpha, official name Gatad2a, MGI:2384585). We found that mp66alpha is essential for development, as mutant embryos die around day 10 of embryogenesis. The gene is not required for normal blastocyst development or for implantation. The phenotype of mutant embryos and the pattern of gene expression in mutants are consistent with a role of mp66alpha in gene silencing. mp66alpha is an essential gene, required for early mouse development. The lethal phenotype supports a role in execution of methylated DNA silencing.

alpha-Hexachlorocyclohexane (alpha-HCH)

Integrated Risk Information System (IRIS)

alpha - Hexachlorocyclohexane ( alpha - HCH ) ; CASRN 319 - 84 - 6 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Ass

Simultaneous quantification of GABAergic 3alpha,5alpha/3alpha,5beta neuroactive steroids in human and rat serum.

PubMed

Porcu, Patrizia; O'Buckley, Todd K; Alward, Sarah E; Marx, Christine E; Shampine, Lawrence J; Girdler, Susan S; Morrow, A Leslie

2009-01-01

The 3alpha,5alpha- and 3alpha,5beta-reduced derivatives of progesterone, deoxycorticosterone, dehydroepiandrosterone and testosterone enhance GABAergic neurotransmission and produce inhibitory neurobehavioral and anti-inflammatory effects. Despite substantial information on the progesterone derivative (3alpha,5alpha)-3-hydroxypregnan-20-one (3alpha,5alpha-THP, allopregnanolone), the physiological significance of the other endogenous GABAergic neuroactive steroids has remained elusive. Here, we describe the validation of a method using gas chromatography-mass spectrometry to simultaneously identify serum levels of the eight 3alpha,5alpha- and 3alpha,5beta-reduced derivatives of progesterone, deoxycorticosterone, dehydroepiandrosterone and testosterone. The method shows specificity, sensitivity and enhanced throughput compared to other methods already available for neuroactive steroid quantification. Administration of pregnenolone to rats and progesterone to women produced selective effects on the 3alpha,5alpha- and 3alpha,5beta-reduced neuroactive steroids, indicating differential regulation of their biosynthetic pathways. Pregnenolone administration increased serum levels of 3alpha,5alpha-THP (+1488%, p<0.001), (3alpha,5alpha)-3,21-dihydroxypregnan-20-one (3alpha,5alpha-THDOC, +205%, p<0.01), (3alpha,5alpha)-3-hydroxyandrostan-17-one (3alpha,5alpha-A, +216%, p<0.001), (3alpha,5alpha,17beta)-androstane-3,17-diol (3alpha,5alpha-A-diol, +190%, p<0.01). (3alpha,5beta)-3-hydroxypregnan-20-one (3alpha,5beta-THP) and (3alpha,5beta)-3-hydroxyandrostan-17-one (3alpha,5beta-A) were not altered, while (3alpha,5beta)-3,21-dihydroxypregnan-20-one (3alpha,5beta-THDOC) and (3alpha,5beta,17beta)-androstane-3,17-diol (3alpha,5beta-A-diol) were increased from undetectable levels to 271+/-100 and 2.4+/-0.9 pg+/-SEM, respectively (5/8 rats). Progesterone administration increased serum levels of 3alpha,5alpha-THP (+1806%, p<0.0001), 3alpha,5beta-THP (+575%, p<0.001), 3alpha,5alpha

Updating the maize karyotype by chromosome DNA sizing.

PubMed

Silva, Jéssica Coutinho; Carvalho, Carlos Roberto; Clarindo, Wellington Ronildo

2018-01-01

The karyotype is a basic concept regarding the genome, fundamentally described by the number and morphological features of all chromosomes. Chromosome class, centromeric index, intra- and interchromosomal asymmetry index, and constriction localization are important in clinical, systematic and evolutionary approaches. In spite of the advances in karyotype characterization made over the last years, new data about the chromosomes can be generated from quantitative methods, such as image cytometry. Therefore, using Zea mays L., this study aimed to update the species' karyotype by supplementing information on chromosome DNA sizing. After adjustment of the procedures, chromosome morphometry and class as well as knob localization enabled describing the Z. mays karyotype. In addition, applying image cytometry, DNA sizing was unprecedentedly measured for the arms and satellite of all chromosomes. This way, unambiguous identification of the chromosome pairs, and hence the assembly of 51 karyograms, were only possible after the DNA sizing of each chromosome, their arms and satellite portions. These accurate, quantitative and reproducible data also enabled determining the distribution and variation of DNA content in each chromosome. From this, a correlation between DNA amount and total chromosome length evidenced that the mean DNA content of chromosome 9 was higher than that of chromosome 8. The chromosomal DNA sizing updated the Z. mays karyotype, providing insights into its dynamic genome with regards to the organization of the ten chromosomes and their respective portions. Considering the results and the relevance of cytogenetics in the current scenario of comparative sequencing and genomics, chromosomal DNA sizing should be incorporated as an additional parameter for karyotype definition. Based on this study, it can be affirmed that cytogenetic approaches go beyond the simple morphological description of chromosomes.

Chemical conversion of corticosteroids to 3 alpha,5 alpha-tetrahydro derivatives. Synthesis of 5 alpha-cortol 3-glucuronides and 5 alpha-cortolone 3-glucuronides.

PubMed

Hosoda, H; Osanai, K; Nambara, T

1991-12-01

The synthesis of the 3-glucuronides of 5 alpha-cortol-20 alpha, 5 alpha-cortolone-20 alpha and their 20 beta-epimers is described. The 5 alpha-cortol 20,21-diacetates (12, 17) and 5 alpha-cortolone 20,21-diacetates (14, 19) were the key intermediates. Sodium borohydride reduction of the carbonyl group at C-20 in 5 alpha-tetrahydrocortisol 3-tert-butyldimethylsilyl ether 17,21-acetonide (8) gave the 20 alpha-hydroxy-acetonide (9). Selective removal of the acetonide ring was successful when the 20 alpha-acetoxy-17 alpha,21-acetonide (10) was treated with 50% acetic acid. Subsequent acetylation with acetic anhydride in pyridine, followed by removal of the protecting group at C-3 in the silyl ether-acetate (11) gave the desired 20 alpha-intermediate (12). The 11-ketone (14) was prepared from 11 by oxidation with pyridinium chlorochromate, followed by desilylation. The 20 beta-acetates (17, 19) were synthesized from 21-acetoxy-3 alpha,11 beta,17 alpha-trihydroxy-5 alpha-pregnan-20-one 3-tert-butyldimethylsilyl ether (15). Introduction of the glucuronyl residue at C-3 was carried out by means of the Koenigs-Knorr reaction.

The CentO satellite confers translational and rotational phasing on cenH3 nucleosomes in rice centromeres.

PubMed

Zhang, Tao; Talbert, Paul B; Zhang, Wenli; Wu, Yufeng; Yang, Zujun; Henikoff, Jorja G; Henikoff, Steven; Jiang, Jiming

2013-12-10

Plant and animal centromeres comprise megabases of highly repeated satellite sequences, yet centromere function can be specified epigenetically on single-copy DNA by the presence of nucleosomes containing a centromere-specific variant of histone H3 (cenH3). We determined the positions of cenH3 nucleosomes in rice (Oryza sativa), which has centromeres composed of both the 155-bp CentO satellite repeat and single-copy non-CentO sequences. We find that cenH3 nucleosomes protect 90-100 bp of DNA from micrococcal nuclease digestion, sufficient for only a single wrap of DNA around the cenH3 nucleosome core. cenH3 nucleosomes are translationally phased with 155-bp periodicity on CentO repeats, but not on non-CentO sequences. CentO repeats have an ∼10-bp periodicity in WW dinucleotides and in micrococcal nuclease cleavage, providing evidence for rotational phasing of cenH3 nucleosomes on CentO and suggesting that satellites evolve for translational and rotational stabilization of centromeric nucleosomes.

Sequence of the cDNA of a human dihydrodiol dehydrogenase isoform (AKR1C2) and tissue distribution of its mRNA.

PubMed Central

Shiraishi, H; Ishikura, S; Matsuura, K; Deyashiki, Y; Ninomiya, M; Sakai, S; Hara, A

1998-01-01

Human liver contains three isoforms (DD1, DD2 and DD4) of dihydrodiol dehydrogenase with 20alpha- or 3alpha-hydroxysteroid dehydrogenase activity; the dehydrogenases belong to the aldo-oxo reductase (AKR) superfamily. cDNA species encoding DD1 and DD4 have been identified. However, four cDNA species with more than 99% sequence identity have been cloned and are compatible with a partial amino acid sequence of DD2. In this study we have isolated a cDNA clone encoding DD2, which was confirmed by comparison of the properties of the recombinant and hepatic enzymes. This cDNA showed differences of one, two, four and five nucleotides from the previously reported four cDNA species for a dehydrogenase of human colon carcinoma HT29 cells, human prostatic 3alpha-hydroxysteroid dehydrogenase, a human liver 3alpha-hydroxysteroid dehydrogenase-like protein and chlordecone reductase-like protein respectively. Expression of mRNA species for the five similar cDNA species in 20 liver samples and 10 other different tissue samples was examined by reverse transcriptase-mediated PCR with specific primers followed by diagnostic restriction with endonucleases. All the tissues expressed only one mRNA species corresponding to the newly identified cDNA for DD2: mRNA transcripts corresponding to the other cDNA species were not detected. We suggest that the new cDNA is derived from the principal gene for DD2, which has been named AKR1C2 by a new nomenclature for the AKR superfamily. It is possible that some of the other cDNA species previously reported are rare allelic variants of this gene. PMID:9716498

Stereoselective Synthesis of [alpha, alpha][superscript ']-Biprolines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vartak, Ashish P.; Young, Jr., Victor G.; Johnson, Rodney L.

2010-11-10

A means to induce dehydrodimerization of Seebach's oxazolidinone (5), the stereochemical outcome of which is entirely temperature dependent, is described. The resultant dimers 3 and 4 are precursors to (R,R)-alpha,alpha'-biproline (1) and meso-alpha,alpha'-biproline (2), respectively. An organohypobromite and an iminium halide are proposed to serve as electrophiles in the reaction with the enolate of 5 to give 3 and 4, respectively.

Novel alpha-galactosidase A mutation in a female with recurrent strokes.

PubMed

Tuttolomondo, Antonino; Duro, Giovanni; Miceli, Salvatore; Di Raimondo, Domenico; Pecoraro, Rosaria; Serio, Antonia; Albeggiani, Giuseppe; Nuzzo, Domenico; Iemolo, Francesco; Pizzo, Federica; Sciarrino, Serafina; Licata, Giuseppe; Pinto, Antonio

2012-11-01

Anderson-Fabry disease (AFD) is an X-linked inborn error of glycosphingolipid catabolism resulting from the deficient activity of the lysosomal exoglycohydrolase, a-galactosidase A. The complete genomic and cDNA sequences of the human alpha-galactosidase A gene have been determined and to date, several disease-causing alpha-galactosidase A mutations have been identified, including missense mutations, small deletions/insertions, splice mutations, and large gene rearrangements We report a case of a 56-year-old woman with recurrent cryptogenic strokes. Ophthalmological examination revealed whorled opacities of the cornea (cornea verticillata) and dilated tortuous conjunctival vessels. She did not show other typical signs of Fabry disease such as acroparesthesias and angiokeratoma. The patient's alpha-galactosidase A activity was 4.13 nmol/mL/h in whole blood. Alpha-galactosidase A gene sequence analysis revealed a heterozygous single nucleotide point mutation at nucleotide c.550T>A in exon 4 in this woman, leading to the p.Tyr184Asn amino acid substitution. Copyright © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Distribution of alpha3, alpha5 and alpha(v) integrin subunits in mature and immature human oocytes.

PubMed

Capmany, G; Mart, M; Santaló, J; Bolton, V N

1998-10-01

The distribution of three integrin subunits, alpha3, alpha5 and alpha(v), in immature and mature human oocytes has been examined using immunofluorescence and confocal microscopy. The results demonstrate that both alpha5 and alpha(v) are present at the germinal vesicle stage, while alpha3 was only detected in oocytes after germinal vesicle breakdown, in metaphase I and II stage oocytes. The cortical concentration of integrin subunits alpha3 and alpha5 is consistent with their localization in the oolemma. In contrast, the homogeneous distribution of alpha(v) throughout the oocyte suggests the existence of cytoplasmic reservoirs of this protein in the oocyte.

[Forced Oscillations of DNA Bases].

PubMed

Yakushevich, L V; Krasnobaeva, L A

2016-01-01

This paper presents the results of the studying of forced angular oscillations of the DNA bases with the help of the mathematical model consisting of two coupled nonlinear differential equations that take into account the effects of dissipation and the influence of an external periodic field. The calculation results are illustrated for sequence of gene encoding interferon alpha 17 (IFNA 17).

Complete nucleotide sequences of okra isolates of Cotton leaf curl Gezira virus and their associated DNA-beta from Niger.

PubMed

Shih, S L; Kumar, S; Tsai, W S; Lee, L M; Green, S K

2009-01-01

Okra (Abelmoschus esculentus) is a major crop in Niger. In the fall of 2007, okra leaf curl disease was observed in Niger and the begomovirus and DNA-beta satellite were found associated with the disease. The complete nucleotide sequences of DNA-A (FJ469626 and FJ469627) and associated DNA-beta satellites (FJ469628 and FJ469629) were determined from two samples. This is the first report of molecular characterization of okra-infecting begomovirus and their associated DNA-beta from Niger. The begomovirus and DNA-beta have been identified as Cotton leaf curl Gezira virus and Cotton leaf curl Gezira betasatellite, respectively, which are reported to also infect okra in Egypt, Mali and Sudan.

The phylogenetic and evolutionary history of a novel alpha-globin-type gene in orangutans (Pongo pygmaeus).

PubMed

Steiper, Michael E; Wolfe, Nathan D; Karesh, William B; Kilbourn, Annelisa M; Bosi, Edwin J; Ruvolo, Maryellen

2006-07-01

The alpha-globin genes are implicated in human resistance to malaria, a disease caused by Plasmodium parasites. This study is the first to analyze DNA sequences from a novel alpha-globin-type gene in orangutans, a species affected by Plasmodium. Phylogenetic methods show that the gene is a duplication of an alpha-globin gene and is located 5' of alpha-2 globin. The alpha-globin-type gene is notable for having four amino acid replacements relative to the orangutan's alpha-1 and alpha-2 globin genes, with no synonymous differences. Pairwise K(a)/K(s) methods and likelihood ratio tests (LRTs) revealed that the evolutionary history of the alpha-globin-type gene has been marked by either neutral or positive evolution, but not purifying selection. A comparative analysis of the amino acid replacements of the alpha-globin-type gene with human hemoglobinopathies and hemoglobin structure showed that two of the four replaced sites are members of the same molecular bond, one that is crucial to the proper functioning of the hemoglobin molecule. This suggested an adaptive evolutionary change. Functionally, this locus may result in a thalassemia-like phenotype in orangutans, possibly as an adaptation to combat Plasmodium.

Extended follow-up of anti-HBe-positive patients with chronic hepatitis B retreated with ribavirin and interferon-alpha.

PubMed

Carreño, V; Rico, M A; Pardo, M; Quiroga, J A

2001-11-01

In a pilot study of combination therapy with ribavirin and IFN alpha conducted in anti-HBe-positive individuals with chronic hepatitis B, 21% of patients achieved a sustained ALT normalization and clearance of hepatitis B virus (HBV) DNA as measured by PCR. The present work has assessed whether these sustained responses are lasting long-term. In addition, IFN gamma levels were tested serially in serum as a measure of the immune system activation during treatment. By extending the post-treatment follow-up period 2 years the occurrence of delayed HBV DNA relapses was observed. A low serum level of IFN gamma was detected during and after treatment. IFN gamma demonstrated a multiphasic time-course: the amount of IFN gamma increased in parallel with reductions in HBV DNA but also with ALT flare-ups. In conclusion, the extended follow-up study of anti-HBe-positive patients after combined treatment with ribavirin and IFN alpha has shown that sustained responses are lasting in 17% patients but also that a late onset HBV DNA relapse may occur.

Rationale and Design of the Genomic Research in Alpha-1 Antitrypsin Deficiency and Sarcoidosis Study. Alpha-1 Protocol.

PubMed

Strange, Charlie; Senior, Robert M; Sciurba, Frank; O'Neal, Scott; Morris, Alison; Wisniewski, Stephen R; Bowler, Russell; Hochheiser, Harry S; Becich, Michael J; Zhang, Yingze; Leader, Joseph K; Methé, Barbara A; Kaminski, Naftali; Sandhaus, Robert A

2015-10-01

Severe deficiency of alpha-1 antitrypsin has a highly variable clinical presentation. The Genomic Research in Alpha-1 Antitrypsin Deficiency and Sarcoidosis α1 Study is a prospective, multicenter, cross-sectional study of adults older than age 35 years with PiZZ or PiMZ alpha-1 antitrypsin genotypes. It is designed to better understand if microbial factors influence this heterogeneity. Clinical symptoms, pulmonary function testing, computed chest tomography, exercise capacity, and bronchoalveolar lavage (BAL) will be used to define chronic obstructive pulmonary disease (COPD) phenotypes that can be studied with an integrated systems biology approach that includes plasma proteomics; mouth, BAL, and stool microbiome and virome analysis; and blood microRNA and blood mononuclear cell RNA and DNA profiling. We will rely on global genome, transcriptome, proteome, and metabolome datasets. Matched cohorts of PiZZ participants on or off alpha-1 antitrypsin augmentation therapy, PiMZ participants not on augmentation therapy, and control participants from the Subpopulations and Intermediate Outcome Measures in COPD Study who match on FEV1 and age will be compared. In the primary analysis, we will determine if the PiZZ individuals on augmentation therapy have a difference in lower respiratory tract microbes identified compared with matched PiZZ individuals who are not on augmentation therapy. By characterizing the microbiome in alpha-1 antitrypsin deficiency (AATD), we hope to define new phenotypes of COPD that explain some of the diversity of clinical presentations. As a unique genetic cause of COPD, AATD may inform typical COPD pathogenesis, and better understanding of it may illuminate the complex interplay between environment and genetics. Although the biologic approaches are hypothesis generating, the results may lead to development of novel biomarkers, better understanding of COPD phenotypes, and development of novel diagnostic and therapeutic trials in AATD and COPD

Rationale and Design of the Genomic Research in Alpha-1 Antitrypsin Deficiency and Sarcoidosis Study. Alpha-1 Protocol

PubMed Central

Senior, Robert M.; Sciurba, Frank; O’Neal, Scott; Morris, Alison; Wisniewski, Stephen R.; Bowler, Russell; Hochheiser, Harry S.; Becich, Michael J.; Zhang, Yingze; Leader, Joseph K.; Methé, Barbara A.; Kaminski, Naftali; Sandhaus, Robert A.

2015-01-01

Severe deficiency of alpha-1 antitrypsin has a highly variable clinical presentation. The Genomic Research in Alpha-1 Antitrypsin Deficiency and Sarcoidosis α1 Study is a prospective, multicenter, cross-sectional study of adults older than age 35 years with PiZZ or PiMZ alpha-1 antitrypsin genotypes. It is designed to better understand if microbial factors influence this heterogeneity. Clinical symptoms, pulmonary function testing, computed chest tomography, exercise capacity, and bronchoalveolar lavage (BAL) will be used to define chronic obstructive pulmonary disease (COPD) phenotypes that can be studied with an integrated systems biology approach that includes plasma proteomics; mouth, BAL, and stool microbiome and virome analysis; and blood microRNA and blood mononuclear cell RNA and DNA profiling. We will rely on global genome, transcriptome, proteome, and metabolome datasets. Matched cohorts of PiZZ participants on or off alpha-1 antitrypsin augmentation therapy, PiMZ participants not on augmentation therapy, and control participants from the Subpopulations and Intermediate Outcome Measures in COPD Study who match on FEV1 and age will be compared. In the primary analysis, we will determine if the PiZZ individuals on augmentation therapy have a difference in lower respiratory tract microbes identified compared with matched PiZZ individuals who are not on augmentation therapy. By characterizing the microbiome in alpha-1 antitrypsin deficiency (AATD), we hope to define new phenotypes of COPD that explain some of the diversity of clinical presentations. As a unique genetic cause of COPD, AATD may inform typical COPD pathogenesis, and better understanding of it may illuminate the complex interplay between environment and genetics. Although the biologic approaches are hypothesis generating, the results may lead to development of novel biomarkers, better understanding of COPD phenotypes, and development of novel diagnostic and therapeutic trials in AATD and COPD

Exploring transmembrane transport through alpha-hemolysin with grid-steered molecular dynamics.

PubMed

Wells, David B; Abramkina, Volha; Aksimentiev, Aleksei

2007-09-28

The transport of biomolecules across cell boundaries is central to cellular function. While structures of many membrane channels are known, the permeation mechanism is known only for a select few. Molecular dynamics (MD) is a computational method that can provide an accurate description of permeation events at the atomic level, which is required for understanding the transport mechanism. However, due to the relatively short time scales accessible to this method, it is of limited utility. Here, we present a method for all-atom simulation of electric field-driven transport of large solutes through membrane channels, which in tens of nanoseconds can provide a realistic account of a permeation event that would require a millisecond simulation using conventional MD. In this method, the average distribution of the electrostatic potential in a membrane channel under a transmembrane bias of interest is determined first from an all-atom MD simulation. This electrostatic potential, defined on a grid, is subsequently applied to a charged solute to steer its permeation through the membrane channel. We apply this method to investigate permeation of DNA strands, DNA hairpins, and alpha-helical peptides through alpha-hemolysin. To test the accuracy of the method, we computed the relative permeation rates of DNA strands having different sequences and global orientations. The results of the G-SMD simulations were found to be in good agreement in experiment.

Peptide-matrix-mediated gene transfer of an oxygen-insensitive hypoxia-inducible factor-1alpha variant for local induction of angiogenesis.

PubMed

Trentin, Diana; Hall, Heike; Wechsler, Sandra; Hubbell, Jeffrey A

2006-02-21

Hypoxia-inducible factor (HIF) constitutes a target in therapeutic angiogenesis. HIF-1alpha functions as a sensor of hypoxia and induces expression of vascular endothelial growth factor (VEGF), which then induces angiogenesis. To explore the potential of HIF-1alpha gene therapy in stimulating wound healing, we delivered a gene encoding a stabilized form of HIF-1alpha, lacking the oxygen-sensitive degradation domain, namely HIF-1alpha deltaODD, by using a previously characterized peptide-based gene delivery vector in fibrin as a surgical matrix. The peptide vector consisted of multiple domains: (i) A cysteine-flanked lysine hexamer provided DNA interactions that were stable extracellularly but destabilized intracellularly after reduction of the formed disulfide bonds. This DNA-binding domain was fused to either (ii) a fibrin-binding peptide for entrapment within the matrix or (iii) a nuclear localization sequence for efficient nuclear targeting. The HIF-1alpha deltaODD gene was expressed and translocated to the nucleus under normoxic conditions, leading to up-regulation of vascular endothelial growth factor (VEGF)-A165 mRNA and protein levels in vitro. When the peptide-DNA nanoparticles entrapped in fibrin matrices were applied to full-thickness dermal wounds in the mouse (10 microg per wound in 30 microl of fibrin), angiogenesis was increased comparably strongly to that induced by VEGF-A165 protein (1.25 microg per wound in 30 microl of fibrin). However, the maturity of the vessels induced by HIF-1alpha deltaODD was significantly higher than that induced by VEGF-A165 protein, as shown by stabilization of the neovessels with smooth muscle. Nonviral, local administration of this potent angiogenesis-inducing gene by using this peptide vector represents a powerful approach in tissue engineering and therapeutic angiogenesis.

Structure of the E2 DNA-binding domain from human papillomavirus serotype 31 at 2.4 A.

PubMed

Bussiere, D E; Kong, X; Egan, D A; Walter, K; Holzman, T F; Lindh, F; Robins, T; Giranda, V L

1998-11-01

The papillomaviruses are a family of small double-stranded DNA viruses which exclusively infect epithelial cells and stimulate the proliferation of those cells. A key protein within the papillomavirus life-cycle is known as the E2 (Early 2) protein and is responsible for regulating viral transcription from all viral promoters as well as for replication of the papillomavirus genome in tandem with another protein known as E1. The E2 protein itself consists of three functional domains: an N-terminal trans-activation domain, a proline-rich linker, and a C-terminal DNA-binding domain. The first crystal structure of the human papillomavirus, serotype 31 (HPV-31), E2 DNA-binding domain has been determined at 2.4 A resolution. The HPV DNA-binding domain monomer consists of two beta-alpha-beta repeats of approximately equal length and is arranged as to have an anti-parallel beta-sheet flanked by the two alpha-helices. The monomers form the functional in vivo dimer by association of the beta-sheets of each monomer so as to form an eight-stranded anti-parallel beta-barrel at the center of the dimer, with the alpha-helices lining the outside of the barrel. The overall structure of HVP-31 E2 DNA-binding domain is similar to both the bovine papillomavirus E2-binding domain and the Epstein-Barr nuclear antigen-1 DNA-binding domain.

Structural analysis of the recognition of the negative regulator NmrA and DNA by the zinc finger from the GATA-type transcription factor AreA.

PubMed

Kotaka, Masayo; Johnson, Christopher; Lamb, Heather K; Hawkins, Alastair R; Ren, Jingshan; Stammers, David K

2008-08-29

Amongst the most common protein motifs in eukaryotes are zinc fingers (ZFs), which, although largely known as DNA binding modules, also can have additional important regulatory roles in forming protein:protein interactions. AreA is a transcriptional activator central to nitrogen metabolism in Aspergillus nidulans. AreA contains a GATA-type ZF that has a competing dual recognition function, binding either DNA or the negative regulator NmrA. We report the crystal structures of three AreA ZF-NmrA complexes including two with bound NAD(+) or NADP(+). The molecular recognition of AreA ZF-NmrA involves binding of the ZF to NmrA via hydrophobic and hydrogen bonding interactions through helices alpha1, alpha6 and alpha11. Comparison with an earlier NMR solution structure of AreA ZF-DNA complex by overlap of the AreA ZFs shows that parts of helices alpha6 and alpha11 of NmrA are positioned close to the GATA motif of the DNA, mimicking the major groove of DNA. The extensive overlap of DNA with NmrA explains their mutually exclusive binding to the AreA ZF. The presence of bound NAD(+)/NADP(+) in the NmrA-AreaA ZF complex, however, causes minimal structural changes. Thus, any regulatory effects on AreA function mediated by the binding of oxidised nicotinamide dinucleotides to NmrA in the NmrA-AreA ZF complex appear not to be modulated via protein conformational rearrangements.

Updating the maize karyotype by chromosome DNA sizing

PubMed Central

2018-01-01

The karyotype is a basic concept regarding the genome, fundamentally described by the number and morphological features of all chromosomes. Chromosome class, centromeric index, intra- and interchromosomal asymmetry index, and constriction localization are important in clinical, systematic and evolutionary approaches. In spite of the advances in karyotype characterization made over the last years, new data about the chromosomes can be generated from quantitative methods, such as image cytometry. Therefore, using Zea mays L., this study aimed to update the species’ karyotype by supplementing information on chromosome DNA sizing. After adjustment of the procedures, chromosome morphometry and class as well as knob localization enabled describing the Z. mays karyotype. In addition, applying image cytometry, DNA sizing was unprecedentedly measured for the arms and satellite of all chromosomes. This way, unambiguous identification of the chromosome pairs, and hence the assembly of 51 karyograms, were only possible after the DNA sizing of each chromosome, their arms and satellite portions. These accurate, quantitative and reproducible data also enabled determining the distribution and variation of DNA content in each chromosome. From this, a correlation between DNA amount and total chromosome length evidenced that the mean DNA content of chromosome 9 was higher than that of chromosome 8. The chromosomal DNA sizing updated the Z. mays karyotype, providing insights into its dynamic genome with regards to the organization of the ten chromosomes and their respective portions. Considering the results and the relevance of cytogenetics in the current scenario of comparative sequencing and genomics, chromosomal DNA sizing should be incorporated as an additional parameter for karyotype definition. Based on this study, it can be affirmed that cytogenetic approaches go beyond the simple morphological description of chromosomes. PMID:29293613

Alpha-tubulin missense mutations correlate with antimicrotubule drug resistance in Eleusine indica.

PubMed Central

Yamamoto, E; Zeng, L; Baird, W V

1998-01-01

Dinitroaniline herbicides are antimicrotubule drugs that bind to tubulins and inhibit polymerization. As a result of repeated application of dinitroaniline herbicides, highly resistant and intermediately resistant biotypes of goosegrass (Eleusine indica) developed in previously wild-type populations. Three alpha-tubulin cDNA classes (designated TUA1, TUA2, and TUA3) were isolated from each biotype. Nucleotide differences between the susceptible and the resistant (R) alpha-tubulins were identified in TUA1 and TUA2. The most significant differences were missense mutations that occurred in TUA1 of the R and intermediately resistant (I) biotypes. Such mutations convert Thr-239 to Ile in the R biotype and Met-268 to Thr in the I biotype. These amino acid substitutions alter hydrophobicity; therefore, they may alter the dinitroaniline binding property of the protein. These mutations were correlated with the dinitroaniline response phenotypes (Drp). Plants homozygous for susceptibility possessed the wild-type TUA1 allele; plants homozygous for resistance possessed the mutant tua1 allele; and plants heterozygous for susceptibility possessed both wild-type and mutant alleles. Thus, we conclude that TUA1 is at the Drp locus. Using polymerase chain reaction primer-introduced restriction analysis, we demonstrated that goosegrass genomic DNA can be diagnosed for Drp alleles. Although not direct proof, these results suggest that a mutation in an alpha-tubulin gene confers resistance to dinitroanilines in goosegrass. PMID:9490751

Alpha-tubulin missense mutations correlate with antimicrotubule drug resistance in Eleusine indica.

PubMed

Yamamoto, E; Zeng, L; Baird, W V

1998-02-01

Dinitroaniline herbicides are antimicrotubule drugs that bind to tubulins and inhibit polymerization. As a result of repeated application of dinitroaniline herbicides, highly resistant and intermediately resistant biotypes of goosegrass (Eleusine indica) developed in previously wild-type populations. Three alpha-tubulin cDNA classes (designated TUA1, TUA2, and TUA3) were isolated from each biotype. Nucleotide differences between the susceptible and the resistant (R) alpha-tubulins were identified in TUA1 and TUA2. The most significant differences were missense mutations that occurred in TUA1 of the R and intermediately resistant (I) biotypes. Such mutations convert Thr-239 to Ile in the R biotype and Met-268 to Thr in the I biotype. These amino acid substitutions alter hydrophobicity; therefore, they may alter the dinitroaniline binding property of the protein. These mutations were correlated with the dinitroaniline response phenotypes (Drp). Plants homozygous for susceptibility possessed the wild-type TUA1 allele; plants homozygous for resistance possessed the mutant tua1 allele; and plants heterozygous for susceptibility possessed both wild-type and mutant alleles. Thus, we conclude that TUA1 is at the Drp locus. Using polymerase chain reaction primer-introduced restriction analysis, we demonstrated that goosegrass genomic DNA can be diagnosed for Drp alleles. Although not direct proof, these results suggest that a mutation in an alpha-tubulin gene confers resistance to dinitroanilines in goosegrass.

A Survey of Geosynchronous Satellite Glints

NASA Astrophysics Data System (ADS)

Vrba, F.; Hutter, D.; Shankland, P.; Armstrong, J.; Schmitt, H.; Hindsley, R.; Divittorio, M.; Benson, J.

Artificial satellites have characteristic diffuse reflected-light signatures as they are illuminated at varying phase angles by the Sun and are viewed at differing orientations by an observer. At times of favorable alignment between the satellite, observer and Sun, specular reflection off of relatively flat surfaces, such as solar panels, can cause brief increases in reflected light of several hundred times that of the nominal diffuse signature. Such events are commonly referred to as "glints". In the case of geosynchronous satellites, favorable glint alignments are due to changes in the Sun-Vehicle-Observer angle which are primarily due to the apparent motion of the Sun as the observer-satellite vector remains nearly stationary. These occur near in time to the vernal and autumnal equinoxes. While the most favorable geosynchronous satellite glint alignments are precluded by the fact that the satellites are at that time most likely to be in Earth shadow, observations of several glints have been reported in the literature. While such studies note the peak brightnesses, durations, and phase angles of individual glints, to our knowledge, no extended study of geosynchronous glint characteristics exists. Beginning with the autumnal equinox glint season of 2007 we have built on our earlier studies using the U.S. Naval Observatory, Flagstaff Station 40-inch Ritchey telescope to provide near-real-time astrometric and photometric information for use by the Navy Prototype Optical Interferometer (NPOI) team in its efforts to obtain interferometric fringes of geosynchronous satellites during a glint episode. The combined observations culminated in successful fringe measurements of DirecTV-9S during the vernal equinox 2008 and 2009 seasons (see Armstrong, et al. 2009, this conference). For our 40-inch telescope observations we used an LN2-cooled 2048x2048 CCD with standard R-band and H-alpha photometric filters, covering an area of the sky of approximately 22x22 arcmin with each

Working the kinks out of nucleosomal DNA

PubMed Central

Olson, Wilma K.; Zhurkin, Victor B.

2011-01-01

Condensation of DNA in the nucleosome takes advantage of its double-helical architecture. The DNA deforms at sites where the base pairs face the histone octamer. The largest so-called kink-and-slide deformations occur in the vicinity of arginines that penetrate the minor groove. Nucleosome structures formed from the 601 positioning sequence differ subtly from those incorporating an AT-rich human α-satellite DNA. Restraints imposed by the histone arginines on the displacement of base pairs can modulate the sequence-dependent deformability of DNA and potentially contribute to the unique features of the different nucleosomes. Steric barriers mimicking constraints found in the nucleosome induce the simulated large-scale rearrangement of canonical B-DNA to kink-and-slide states. The pathway to these states shows non-harmonic behavior consistent with bending profiles inferred from AFM measurements. PMID:21482100

Fibrinogen Lincoln: a new truncated alpha chain variant with delayed clotting.

PubMed

Ridgway, H J; Brennan, S O; Gibbons, S; George, P M

1996-04-01

A patient referred for preoperative investigation of prolonged bleeding and easy bruising was found to have increased thrombin and reptilase times; however, the thrombin catalysed release of fibrinopeptides A and B was normal. Analysis of five other family members, spanning three generations, indicated that three had a similar defect and suggested autosomal dominant inheritance. Non-reducing SDS-PAGE of purified fibrinogen from affected individuals showed that the 340 kD form of their fibrinogen ran as a doublet. SSCP (single-stranded conformational polymorphism) analysis of exon 5 of the A alpha gene, which encodes the C-terminal half of the chain, confirmed the presence of a mutation. Cycle sequencing of PCR amplified DNA revealed a 13 base pair deletion (nt 4758-4770), resulting in a frame-shift at Ala 475, which translates as four new amino acids before terminating at a new stop codon (-476His-Cys-Leu-Ala-Stop). The presence of a circulating truncated A alpha chain was confirmed when SDS-PAGE gels were probed with an alpha chain specific antisera; which showed that the variant A alpha chain comigrated with gamma chains. The truncation results in a variant A alpha chain with a deletion of 131 amino acids (480-610), and four new amino acids at the C-terminal.

Weighing the Low-Redshift Lyman-alpha Forest

NASA Technical Reports Server (NTRS)

Shull, Mike

2005-01-01

In 2003-2004, our FUSE research group prepared several major surveys of the amount of baryonic matter in the intergalactic medium (IGM), using the Far Ultraviolet Spectroscopic Explorer (FUSE) satellite. These surveys include measurements of the Lyman-alpha absorption line of neutral hydrogen (H I), the far-ultraviolet (1032,1038 Angstrom) doublet of highly ionized oxygen ( O VI), the higher Lyman-series lines (Ly-beta, Ly-gamma, etc) of H I, and the 977 Angstrom line of c III. As an overview, our FUSE spectroscopic studies, taken together with data from the Hubble Space Telescope, show that approximately 30% of the normal matter is contained in intergalactic hydrogen gas clouds (the Lyman-alpha forest). Another 5-10% resides in hotter gas at temperatures of 10(exp 5) to 10(exp 6) K, visible in 0 VI and C III absorption. Along with the matter attributed to galaxies, we have now accounted for approximately HALF of all the baryonic matter in the universe. Where is the other half? That matter my exist in even hotter gas, invisible through the ultraviolet absorption lines, but perhaps detectable through X-ray absorption lines of more highly ionized oxygen and neon.

Behavior of a cloned murine interferon alpha/beta receptor expressed in homospecific or heterospecific background.

PubMed

Uzé, G; Lutfalla, G; Bandu, M T; Proudhon, D; Mogensen, K E

1992-05-15

A murine interferon (IFN) alpha/beta receptor was cloned from the IFN-sensitive L1210 cell line on the basis of its homology with the human receptor. A combination of methods that includes the screening of random-primed and oligo(dT)-primed cDNA libraries and polymerase chain reactions with a single-side specificity was used. At the amino acid level, the murine IFN-alpha/beta shows 46% identity with its human counterpart. Both human WISH cells presenting a low sensitivity to mouse IFN and a murine L1210 mutant subline that does not express the receptor have been stably transfected with the murine IFN-alpha/beta receptor. Whereas transfected human cells became sensitive to a limited number of mouse IFN-alpha/beta subtypes, the transfected murine L1210 mutant was found to be fully complemented and became sensitive to all mouse IFN-alpha/beta subtypes tested, including those that were not active on transfected human cells. These results strongly suggest that the receptor described here is implicated in the mediation of the activities of all murine IFN-alpha/beta subtypes.

Orthopositronium Lifetime: Analytic Results in O({alpha}) and O({alpha}{sup 3}ln{alpha})

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kniehl, Bernd A.; Kotikov, Anatoly V.; Veretin, Oleg L.

2008-11-07

We present the O({alpha}) and O({alpha}{sup 3}ln{alpha}) corrections to the total decay width of orthopositronium in closed analytic form, in terms of basic irrational numbers, which can be evaluated numerically to arbitrary precision.

Mechanisms involved in regulating DNA replication origins during the cell cycle and in response to DNA damage.

PubMed Central

Early, Anne; Drury, Lucy S; Diffley, John F X

2004-01-01

Replication origins in eukaryotic cells never fire more than once in a given S phase. Here, we summarize the role of cyclin-dependent kinases in limiting DNA replication origin usage to once per cell cycle in the budding yeast Saccharomyces cerevisiae. We have examined the role of different cyclins in the phosphorylation and regulation of several replication/regulatory factors including Cdc6, Sic1, ORC and DNA polymerase alpha-primase. In addition to being regulated by the cell cycle machinery, replication origins are also regulated by the genome integrity checkpoint kinases, Mec1 and Rad53. In response to DNA damage or drugs which interfere with the progression of replication forks, the activation of late-firing replication origins is inhibited. There is evidence indicating that the temporal programme of origin firing depends upon the local histone acetylation state. We have attempted to test the possibility that checkpoint regulation of late-origin firing operates through the regulation of the acetylation state. We found that overexpression of the essential histone acetylase, Esal, cannot override checkpoint regulation of origin firing. We have also constructed a temperature-sensitive esa1 mutant. This mutant is unable to resume cell cycle progression after alpha-factor arrest. This can be overcome by overexpression of the G1 cyclin, Cln2, revealing a novel role for Esal in regulating Start. PMID:15065654

Effects of alpha-tocopherol treatment on newborn rat lung development and injury in hyperoxia.

PubMed

Bucher, J R; Roberts, R J

1982-01-01

The efficacy of alpha-tocopherol treatment to influence the pattern or extent of lung injury resulting during exposure of newborn rats to hyperoxia was assessed following six-day exposures to FIO2 0.21, 0.4, and greater than 0.95. Alpha-Tocopherol treatment was found incapable of preventing the developmental arrest of the lung that occurs during hyperoxic exposure, shown by assessments of wet lung weights, lung DNA, lung volumes, and the progress of secondary septal and capillary development. However alpha-tocopherol treatment was found effective in preventing the hyperoxic-induced lessening of lung compliance and in preventing the deterioration of gas exchange capacity in the lung of the hyperoxic-exposed newborn rat. These findings suggest alpha-tocopherol treatment may not be capable of preventing major alterations in lung morphology in infants with chronic lung disease may be lessened by preserving gas exchange capabilities.

The human interleukin-1 alpha gene is located on the long arm of chromosome 2 at band q13.

PubMed

Lafage, M; Maroc, N; Dubreuil, P; de Waal Malefijt, R; Pébusque, M J; Carcassonne, Y; Mannoni, P

1989-01-01

Interleukin-1 alpha (IL-1 alpha) and interleukin-1 beta (IL-1 beta) are two biochemically distinct, but distantly related, polypeptidic cytokines that play a key role in inflammation, immunologic reactions, and tissue repair. Recently, it has been shown that IL-1 alpha is identical to hematopoietin 1, which was described as a hematopoietic growth factor acting on early progenitor cells in synergy with other hematopoietic growth factors. In this report we discuss our use of in situ hybridization on human prometaphase cells with a human IL-1 alpha cDNA probe to localize the human IL-1 alpha gene on the proximal part of the long arm of chromosome 2 at band q13, in the same chromosomal region as the IL-1 beta gene.

Replication, checkpoint suppression and structure of centromeric DNA

PubMed Central

Romeo, Francesco; Costanzo, Vincenzo

2016-01-01

ABSTRACT Human centromeres contain large amounts of repetitive DNA sequences known as α satellite DNA, which can be difficult to replicate and whose functional role is unclear. Recently, we have characterized protein composition, structural organization and checkpoint response to stalled replication forks of centromeric chromatin reconstituted in Xenopus laevis egg extract. We showed that centromeric DNA has high affinity for SMC2-4 subunits of condensins and for CENP-A, it is enriched for DNA repair factors and suppresses the ATR checkpoint to ensure its efficient replication. We also showed that centromeric chromatin forms condensins enriched and topologically constrained DNA loops, which likely contribute to the overall structure of the centromere. These findings have important implications on how chromosomes are organized and genome stability is maintained in mammalian cells. PMID:27893298

TRF2 and apollo cooperate with topoisomerase 2alpha to protect human telomeres from replicative damage.

PubMed

Ye, Jing; Lenain, Christelle; Bauwens, Serge; Rizzo, Angela; Saint-Léger, Adelaïde; Poulet, Anaïs; Benarroch, Delphine; Magdinier, Frédérique; Morere, Julia; Amiard, Simon; Verhoeyen, Els; Britton, Sébastien; Calsou, Patrick; Salles, Bernard; Bizard, Anna; Nadal, Marc; Salvati, Erica; Sabatier, Laure; Wu, Yunlin; Biroccio, Annamaria; Londoño-Vallejo, Arturo; Giraud-Panis, Marie-Josèphe; Gilson, Eric

2010-07-23

Human telomeres are protected from DNA damage by a nucleoprotein complex that includes the repeat-binding factor TRF2. Here, we report that TRF2 regulates the 5' exonuclease activity of its binding partner, Apollo, a member of the metallo-beta-lactamase family that is required for telomere integrity during S phase. TRF2 and Apollo also suppress damage to engineered interstitial telomere repeat tracts that were inserted far away from chromosome ends. Genetic data indicate that DNA topoisomerase 2alpha acts in the same pathway of telomere protection as TRF2 and Apollo. Moreover, TRF2, which binds preferentially to positively supercoiled DNA substrates, together with Apollo, negatively regulates the amount of TOP1, TOP2alpha, and TOP2beta at telomeres. Our data are consistent with a model in which TRF2 and Apollo relieve topological stress during telomere replication. Our work also suggests that cellular senescence may be caused by topological problems that occur during the replication of the inner portion of telomeres. Copyright 2010 Elsevier Inc. All rights reserved.

Improved synthesis of 3 alpha, 7 alpha, 12 alpha, 24 = xi-tetrahydroxy-5 beta-cholestan-26-oic acid.

PubMed

Batta, A K; Tint, G S; Dayal, B; Shefer, S; Salen, G

1982-06-01

This paper describes three simple and short methods for the conversion of cholic acid into cholylaldehyde with protected hydroxyl groups. The first method involves lithium aluminum hydride reduction of the tetrahydropyranyl ether of methyl cholate and oxidation of the resulting primary alcohol with pyridinium chlorochromate. The second method employs diborane for the reduction of the -COOH group to the -CH2OH group, while the third method involves the reduction of 3 alpha, 7 alpha, 12 alpha-triformyloxy-5 beta-cholan-24-oic acid (as the acid chloride) directly into 3 alpha, 7 alpha, 12 alpha-triformyloxy-5 beta-cholan-24-al with TMA-ferride (tetramethylammonium hydridoirontetracarbonyl). The aldehyde obtained by any of the above methods underwent smooth Reformatsky reaction with ethyl alpha-bromopropionate to yield 3 alpha, 7 alpha, 12 alpha, 24 xi-tetrahydroxy-5 beta-cholestan-26-oic acid.

Structure-activity analysis of synthetic alpha-thrombin-receptor-activating peptides.

PubMed

Van Obberghen-Schilling, E; Rasmussen, U B; Vouret-Craviari, V; Lentes, K U; Pavirani, A; Pouysségur, J

1993-06-15

alpha-Thrombin stimulates G-protein-coupled effectors leading to secretion and aggregation in human platelets, and to a mitogenic response in CCL39 hamster fibroblasts. alpha-Thrombin receptors can be activated by synthetic peptides corresponding to the receptor sequence starting with serine-42, at the proposed cleavage site. We have previously determined that the agonist domain of receptor-activating peptides resides within the five N-terminal residues [Vouret-Craviari, Van Obberghen-Schilling, Rasmussen, Pavirani, Lecocq and Pouysségur (1992) Mol. Biol. Cell. 3, 95-102], although the 7-residue peptide (SFFLRNP) corresponding to the hamster alpha-thrombin receptor was 10 times more potent than the 5-residue peptide for activation of human platelets. In the present study we have analysed the role of individual amino acids in receptor activation by using a series of modified hexa- or hepta-peptides derived from the human alpha-thrombin-receptor sequence. Cellular events examined here include phospholipase C activation, adenylyl cyclase inhibition and DNA synthesis stimulation in non-transformed CCL39 fibroblasts and a tumorigenic variant of that line (A71 cells). Modification of the peptide sequence had similar functional consequence for each of the assays described, indicating that either a unique receptor or pharmacologically indistinguishable receptor subtypes activate distinct G-protein signalling pathways. Furthermore, we found that: (1) the N-terminal serine can be replaced by small or intermediately sized amino acids (+/- hydroxyl groups) without loss of activity. However, its replacement by an aromatic side-chain or omission of the N-terminal amino group severely reduces activity. (2) An aromatic side-chain on the penultimate N-terminal residue appears to play a critical role since phenylalanine in this position can be substituted by tyrosine without complete loss of activity whereas an alanine in its place is not tolerated. (3) Deletion of the first

Structure-activity analysis of synthetic alpha-thrombin-receptor-activating peptides.

PubMed Central

Van Obberghen-Schilling, E; Rasmussen, U B; Vouret-Craviari, V; Lentes, K U; Pavirani, A; Pouysségur, J

1993-01-01

alpha-Thrombin stimulates G-protein-coupled effectors leading to secretion and aggregation in human platelets, and to a mitogenic response in CCL39 hamster fibroblasts. alpha-Thrombin receptors can be activated by synthetic peptides corresponding to the receptor sequence starting with serine-42, at the proposed cleavage site. We have previously determined that the agonist domain of receptor-activating peptides resides within the five N-terminal residues [Vouret-Craviari, Van Obberghen-Schilling, Rasmussen, Pavirani, Lecocq and Pouysségur (1992) Mol. Biol. Cell. 3, 95-102], although the 7-residue peptide (SFFLRNP) corresponding to the hamster alpha-thrombin receptor was 10 times more potent than the 5-residue peptide for activation of human platelets. In the present study we have analysed the role of individual amino acids in receptor activation by using a series of modified hexa- or hepta-peptides derived from the human alpha-thrombin-receptor sequence. Cellular events examined here include phospholipase C activation, adenylyl cyclase inhibition and DNA synthesis stimulation in non-transformed CCL39 fibroblasts and a tumorigenic variant of that line (A71 cells). Modification of the peptide sequence had similar functional consequence for each of the assays described, indicating that either a unique receptor or pharmacologically indistinguishable receptor subtypes activate distinct G-protein signalling pathways. Furthermore, we found that: (1) the N-terminal serine can be replaced by small or intermediately sized amino acids (+/- hydroxyl groups) without loss of activity. However, its replacement by an aromatic side-chain or omission of the N-terminal amino group severely reduces activity. (2) An aromatic side-chain on the penultimate N-terminal residue appears to play a critical role since phenylalanine in this position can be substituted by tyrosine without complete loss of activity whereas an alanine in its place is not tolerated. (3) Deletion of the first

Repetitive sequences in plant nuclear DNA: types, distribution, evolution and function.

PubMed

Mehrotra, Shweta; Goyal, Vinod

2014-08-01

Repetitive DNA sequences are a major component of eukaryotic genomes and may account for up to 90% of the genome size. They can be divided into minisatellite, microsatellite and satellite sequences. Satellite DNA sequences are considered to be a fast-evolving component of eukaryotic genomes, comprising tandemly-arrayed, highly-repetitive and highly-conserved monomer sequences. The monomer unit of satellite DNA is 150-400 base pairs (bp) in length. Repetitive sequences may be species- or genus-specific, and may be centromeric or subtelomeric in nature. They exhibit cohesive and concerted evolution caused by molecular drive, leading to high sequence homogeneity. Repetitive sequences accumulate variations in sequence and copy number during evolution, hence they are important tools for taxonomic and phylogenetic studies, and are known as "tuning knobs" in the evolution. Therefore, knowledge of repetitive sequences assists our understanding of the organization, evolution and behavior of eukaryotic genomes. Repetitive sequences have cytoplasmic, cellular and developmental effects and play a role in chromosomal recombination. In the post-genomics era, with the introduction of next-generation sequencing technology, it is possible to evaluate complex genomes for analyzing repetitive sequences and deciphering the yet unknown functional potential of repetitive sequences. Copyright © 2014 The Authors. Production and hosting by Elsevier Ltd.. All rights reserved.

Detection of benzo[a]pyrene diol epoxide-DNA adducts in peripheral blood lymphocytes and antibodies to the adducts in serum from coke oven workers.

PubMed Central

Harris, C C; Vahakangas, K; Newman, M J; Trivers, G E; Shamsuddin, A; Sinopoli, N; Mann, D L; Wright, W E

1985-01-01

Coke oven workers are exposed to high levels of carcinogenic polycyclic aromatic hydrocarbons, including benzo[a]pyrene (B[a]P), and are at increased risk of lung cancer. Since B[a]P is enzymatically activated to 7 beta,8 alpha-dihydroxy(9 alpha, 10 alpha)epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]PDE) that forms adducts with DNA, the presence of these adducts was measured in DNA from peripheral blood lymphocytes by synchronous fluorescence spectrophotometry and enzyme radioimmunoassay. Approximately two-thirds of the workers had detectable levels of B[a]PDE-DNA adducts. Antibodies to the DNA adducts were also found in the serum of 27% of the workers. B[a]PDE-DNA adducts were not detectable in lymphocytes and antibodies to the adducts were not detected in sera from a control group of nonsmoking laboratory workers. DNA adducts and/or antibodies to the adducts indicate exposure to B[a]P and its metabolic activation to the carcinogenic metabolite that covalently binds to and damages DNA. Detection of adducts and antibodies to them may also be useful as internal dosimeters of the pathobiological effective doses of chemical carcinogens. PMID:2413443

Ca2+ permeability through rat cloned alpha9-containing nicotinic acetylcholine receptors.

PubMed

Fucile, Sergio; Sucapane, Antonietta; Eusebi, Fabrizio

2006-04-01

We investigated the functional properties of rat alpha9 and alpha9alpha10 nicotinic acetylcholine receptors (nAChRs) expressed by transient transfection in the rat GH4C1 cell line, using both Ca(2+) imaging and whole-cell recording. Acute applications of ACh generated short-delay fast-rising and quick-decaying Ca(2+) transients, suppressed in Ca(2+)-free medium and invariably accompanied by the activation of whole-cell inward currents. The mean amplitude of ACh-induced currents was as small as -16 pA in alpha9 subunit cDNA-transfected GH4C1 cells (alpha9-GH4C1), while they were much larger (range: -150 to -300 pA) in alpha9alpha10 subunit cDNAs-transfected GH4C1 cells (alpha9alpha10-GH4C1). Currents were not activated by nicotine, were blocked by methyllycaconitine and were ACh concentration-dependent. Because the Ca(2+) permeability of alpha9-containing nAChRs has been estimated in immortalized cochlear UB/OC-2 mouse cells, we also characterized the ACh-induced responses in these cells. Unlike alpha9- and alpha9alpha10-GH4C1 cells, UB/OC-2 cells responded to ACh with both long-delay methyllycaconitine-insensitive whole-cell currents and long-lasting Ca(2+) transients, the latter being detected in the absence of Ca(2+) in the extracellular medium and being suppressed by the Ca(2+)-ATPase inhibitor thapsigargin, known to deplete IP(3)-sensitive stores. These results indicated the involvement of muscarinic nAChRs and the lack of functional ACh-gated receptor channels in UB/OC-2 cells. Thus, we measured the fractional Ca(2+) current (P(f), i.e. the percentage of total current carried by Ca(2+) ions) in alpha9alpha10-GH4C1, obtaining a P(f) value of 22 +/- 4%; this is the largest value estimated to date for a ligand-gated receptor channel. The physiological role played by Ca(2+) entry through alpha9-containing nAChRs gated by ACh is discussed.

Complete Genome Viral Phylogenies Suggests the Concerted Evolution of Regulatory Cores and Accessory Satellites

PubMed Central

Zanotto, Paolo Marinho de Andrade; Krakauer, David C.

2008-01-01

We consider the concerted evolution of viral genomes in four families of DNA viruses. Given the high rate of horizontal gene transfer among viruses and their hosts, it is an open question as to how representative particular genes are of the evolutionary history of the complete genome. To address the concerted evolution of viral genes, we compared genomic evolution across four distinct, extant viral families. For all four viral families we constructed DNA-dependent DNA polymerase-based (DdDp) phylogenies and in addition, whole genome sequence, as quantitative descriptions of inter-genome relationships. We found that the history of the polymerase gene was highly predictive of the history of the genome as a whole, which we explain in terms of repeated, co-divergence events of the core DdDp gene accompanied by a number of satellite, accessory genetic loci. We also found that the rate of gene gain in baculovirus and poxviruses proceeds significantly more quickly than the rate of gene loss and that there is convergent acquisition of satellite functions promoting contextual adaptation when distinct viral families infect related hosts. The congruence of the genome and polymerase trees suggests that a large set of viral genes, including polymerase, derive from a phylogenetically conserved core of genes of host origin, secondarily reinforced by gene acquisition from common hosts or co-infecting viruses within the host. A single viral genome can be thought of as a mutualistic network, with the core genes acting as an effective host and the satellite genes as effective symbionts. Larger virus genomes show a greater departure from linkage equilibrium between core and satellites functions. PMID:18941535

Effects of alpha lipoic acid on acrylamide-induced hepatotoxicity in rats.

PubMed

Al-Qahtani, F A; Arafah, M; Sharma, B; Siddiqi, N J

2017-07-31

Acrylamide (ACR) is a neurotoxicant, reproductive toxicant, and carcinogen in animal species.  It is used in many industries and has been found to form naturally in foods cooked at high temperatures. Alpha-lipoic acid (ALA) is a naturally occurring antioxidant whose therapeutic effect has been related to its antioxidant activity.  This study was carried out to study the protective effect of alpha lipoic acid on acrylamide induced perturbations in rat liver.  Four groups of rats were studied viz., control rats, acrylamide treated rats, alpha lipoic acid treated rats, and alpha lipoic acid plus acrylamide treated rats. ACR and ALA treatment alone and together caused a signifi-cant increase in hepatic reduced glutathione content while a decrease in hepatic ascorbic content was observed when compared to control group.  ALA pretreatment of acrylamide exposed rats caused no a signifi-cant alteration in superoxide dismutase activity but resulted in a tendency towards restoration of glutathione peroxidase and catalase activity to near normal levels.  Gel electrophoresis showed fragmentation of DNA in the treated groups.  The dose of ALA used in the present study afforded partial restoration of oxidative indices altered by ACR in rat liver.

Non-B-Form DNA Is Enriched at Centromeres

PubMed Central

Henikoff, Steven

2018-01-01

Abstract Animal and plant centromeres are embedded in repetitive “satellite” DNA, but are thought to be epigenetically specified. To define genetic characteristics of centromeres, we surveyed satellite DNA from diverse eukaryotes and identified variation in <10-bp dyad symmetries predicted to adopt non-B-form conformations. Organisms lacking centromeric dyad symmetries had binding sites for sequence-specific DNA-binding proteins with DNA-bending activity. For example, human and mouse centromeres are depleted for dyad symmetries, but are enriched for non-B-form DNA and are associated with binding sites for the conserved DNA-binding protein CENP-B, which is required for artificial centromere function but is paradoxically nonessential. We also detected dyad symmetries and predicted non-B-form DNA structures at neocentromeres, which form at ectopic loci. We propose that centromeres form at non-B-form DNA because of dyad symmetries or are strengthened by sequence-specific DNA binding proteins. This may resolve the CENP-B paradox and provide a general basis for centromere specification. PMID:29365169

Murine recessive hereditary spherocytosis, sph/sph, is caused by a mutation in the erythroid alpha-spectrin gene.

PubMed

Wandersee, N J; Birkenmeier, C S; Gifford, E J; Mohandas, N; Barker, J E

2000-01-01

Spectrin, a heterodimer of alpha- and beta-subunits, is the major protein component of the red blood cell membrane skeleton. The mouse mutation, sph, causes an alpha-spectrin-deficient hereditary spherocytosis with the severe phenotype typical of recessive hereditary spherocytosis in humans. The sph mutation maps to the erythroid alpha-spectrin locus, Spna1, on Chromosome 1. Scanning electron microscopy, osmotic gradient ektacytometry, cDNA cloning, RT-PCR, nucleic acid sequencing, and Northern blot analyses were used to characterize the wild type and sph alleles of the Spna1 locus. Our results confirm the spherocytic nature of sph/sph red blood cells and document a mild spherocytic transition in the +/sph heterozygotes. Sequencing of the full length coding region of the Spna1 wild type allele from the C57BL/6J strain of mice reveals a 2414 residue deduced amino acid sequence that shows the typical 106-amino-acid repeat structure previously described for other members of the spectrin protein family. Sequence analysis of RT-PCR clones from sph/sph alpha-spectrin mRNA identified a single base deletion in repeat 5 that would cause a frame shift and premature termination of the protein. This deletion was confirmed in sph/sph genomic DNA. Northern blot analyses of the distribution of Spna1 mRNA in non-erythroid tissues detects the expression of 8, 2.5 and 2.0 kb transcripts in adult heart. These results predict the heart as an additional site where alpha-spectrin mutations may produce a phenotype and raise the possibility that a novel functional class of small alpha-spectrin isoforms may exist.

Problem-Solving Test: Analysis of DNA Damage Recognizing Proteins in Yeast and Human Cells

ERIC Educational Resources Information Center

Szeberenyi, Jozsef

2013-01-01

The experiment described in this test was aimed at identifying DNA repair proteins in human and yeast cells. Terms to be familiar with before you start to solve the test: DNA repair, germline mutation, somatic mutation, inherited disease, cancer, restriction endonuclease, radioactive labeling, [alpha-[superscript 32]P]ATP, [gamma-[superscript…

Nuclear magnetic resonance-based model of a TF1/HmU-DNA complex.

PubMed

Silva, M V; Pasternack, L B; Kearns, D R

1997-12-15

Transcription factor 1 (TF1), a type II DNA-binding protein encoded by the Bacillus subtilis bacteriophage SPO1, has the capacity for sequence-selective DNA binding and a preference for 5-hydroxymethyl-2'-deoxyuridine (HmU)-containing DNA. In NMR studies of the TF1/HmU-DNA complex, intermolecular NOEs indicate that the flexible beta-ribbon and C-terminal alpha-helix are involved in the DNA-binding site of TF1, placing it in the beta-sheet category of DNA-binding proteins proposed to bind by wrapping two beta-ribbon "arms" around the DNA. Intermolecular and intramolecular NOEs were used to generate an energy-minimized model of the protein-DNA complex in which both DNA bending and protein structure changes are evident.

Effect of long-term exposure to mobile phone radiation on alpha-Int1 gene sequence of Candida albicans.

PubMed

Shahin-Jafari, Ariyo; Bayat, Mansour; Shahhosseiny, Mohammad Hassan; Tajik, Parviz; Roudbar-Mohammadi, Shahla

2016-05-01

Over the last decade, communication industries have witnessed a tremendous expansion, while, the biological effects of electromagnetic waves have not been fully elucidated. Current study aimed at evaluating the mutagenic effect of long-term exposure to 900-MHz radiation on alpha-Int1 gene sequences of Candida albicans. A standard 900 MHz radiation generator was used for radiation. 10 ml volumes from a stock suspension of C. albicans were transferred into 10 polystyrene tubes. Five tubes were exposed at 4 °C to a fixed magnitude of radiation with different time periods of 10, 70, 210, 350 and 490 h. The other 5 tubes were kept far enough from radiation. The samples underwent genomic DNA extraction. PCR amplification of alpha-Int1 gene sequence was done using one set of primers. PCR products were resolved using agarose gel electrophoresis and the nucleotide sequences were determined. All samples showed a clear electrophoretic band around 441 bp and further sequencing revealed the amplified DNA segments are related to alpha-Int1 gene of the yeast. No mutations in the gene were seen in radiation exposed samples. Long-term exposure of the yeast to mobile phone radiation under the above mentioned conditions had no mutagenic effect on alpha-Int1 gene sequence.

A Gas-Poor Planetesimal Feeding Model for the Formation of Giant Planet Satellite Systems: Consequences for the Atmosphere of Titan

NASA Technical Reports Server (NTRS)

Estrada, P. R.; Mosqueira, I.

2005-01-01

Given our presently inadequate understanding of the turbulent state of the solar and planetary nebulae, we believe the way to make progress in satellite formation is to consider two end member models that avoid over-reliance on specific choices of the turbulence (alpha), which is essentially a free parameter. The first end member model postulates turbulence decay once giant planet accretion ends. If so, Keplerian disks must eventually pass through the quiescent phases, so that the survival of satellites (and planets) ultimately hinges on gap-opening. In this scenario, the criterion for gap-opening itself sets the value for the gas surface density of the satellite disk.

Comparative Analysis of Repetitive DNA between the Main Vectors of Chagas Disease: Triatoma infestans and Rhodnius prolixus.

PubMed

Pita, Sebastián; Mora, Pablo; Vela, Jesús; Palomeque, Teresa; Sánchez, Antonio; Panzera, Francisco; Lorite, Pedro

2018-04-24

Chagas disease or American trypanosomiasis affects six to seven million people worldwide, mostly in Latin America. This disease is transmitted by hematophagous insects known as "kissing bugs" (Hemiptera, Triatominae), with Triatoma infestans and Rhodnius prolixus being the two most important vector species. Despite the fact that both species present the same diploid chromosome number (2 n = 22), they have remarkable differences in their total DNA content, chromosome structure and genome organization. Variations in the DNA genome size are expected to be due to differences in the amount of repetitive DNA sequences. The T. infestans genome-wide analysis revealed the existence of 42 satellite DNA families. BLAST searches of these sequences against the R. prolixus genome assembly revealed that only four of these satellite DNA families are shared between both species, suggesting a great differentiation between the Triatoma and Rhodnius genomes. Fluorescence in situ hybridization (FISH) location of these repetitive DNAs in both species showed that they are dispersed on the euchromatic regions of all autosomes and the X chromosome. Regarding the Y chromosome, these common satellite DNAs are absent in T. infestans but they are present in the R. prolixus Y chromosome. These results support a different origin and/or evolution in the Y chromosome of both species.

Anticonvulsant properties of alpha, gamma, and alpha, gamma-substituted gamma-butyrolactones.

PubMed

Klunk, W E; Covey, D F; Ferrendelli, J A

1982-09-01

Derivatives of gamma-butyrolactone (GBL) substituted on the alpha- and/or gamma-positions were synthesized and tested for their effects on behavior in mice, on the electroencephalographs and blood pressure of paralyzed-ventilated guinea pigs, and on electrical activity of incubated hippocampal slices. Several compounds, including alpha-ethyl-alpha-methyl GBL (alpha-EMGBL), alpha, alpha-dimethyl GBL, alpha, gamma-diethyl-alpha, gamma-dimethyl GBL, and gamma-ethyl-gamma-methyl GBL, prevented seizures induced by pentylenetetrazol, beta-ethyl-beta-methyl-gamma-butyrolactone (beta-EMGBL), picrotoxin, or all three compounds in mice and guinea pigs but had no effect on seizures induced by maximal electroshock or bicuculline. Neither gamma-hydroxybutyrate (GHB) nor alpha-isopropylidine GBL had any anticonvulsant activity. The anticonvulsant alpha-substituted compounds had a potent hypotensive effect and antagonized the hypertensive effect of beta-EMGBL, alpha-EMGBL was tested in incubated hippocampal slices and was found to depress basal activity and antagonize excitation induced by beta-EMGBL. These results demonstrate that alpha-alkyl-substituted GBL and, to a lesser extent, gamma-substituted derivatives are anticonvulsant agents and that their effects are strikingly different from those of GHB or beta-alkyl-substituted GBLs, which are epileptogenic. Possibly beta- and alpha-substituted GBLs act at the same site as agonists and antagonists, respectively.

The phase behavior of cationic lipid-DNA complexes.

PubMed Central

May, S; Harries, D; Ben-Shaul, A

2000-01-01

We present a theoretical analysis of the phase behavior of solutions containing DNA, cationic lipids, and nonionic (helper) lipids. Our model allows for five possible structures, treated as incompressible macroscopic phases: two lipid-DNA composite (lipoplex) phases, namely, the lamellar (L(alpha)(C)) and hexagonal (H(II)(C)) complexes; two binary (cationic/neutral) lipid phases, that is, the bilayer (L(alpha)) and inverse-hexagonal (H(II)) structures, and uncomplexed DNA. The free energy of the four lipid-containing phases is expressed as a sum of composition-dependent electrostatic, elastic, and mixing terms. The electrostatic free energies of all phases are calculated based on Poisson-Boltzmann theory. The phase diagram of the system is evaluated by minimizing the total free energy of the three-component mixture with respect to all the compositional degrees of freedom. We show that the phase behavior, in particular the preferred lipid-DNA complex geometry, is governed by a subtle interplay between the electrostatic, elastic, and mixing terms, which depend, in turn, on the lipid composition and lipid/DNA ratio. Detailed calculations are presented for three prototypical systems, exhibiting markedly different phase behaviors. The simplest mixture corresponds to a rigid planar membrane as the lipid source, in which case, only lamellar complexes appear in solution. When the membranes are "soft" (i.e., low bending modulus) the system exhibits the formation of both lamellar and hexagonal complexes, sometimes coexisting with each other, and with pure lipid or DNA phases. The last system corresponds to a lipid mixture involving helper lipids with strong propensity toward the inverse-hexagonal phase. Here, again, the phase diagram is rather complex, revealing a multitude of phase transitions and coexistences. Lamellar and hexagonal complexes appear, sometimes together, in different regions of the phase diagram. PMID:10733951

Hepatic DNA adduct dosimetry in rats fed tamoxifen: a comparison of methods.

PubMed

Schild, Laura J; Phillips, David H; Osborne, Martin R; Hewer, Alan; Beland, Frederick A; Churchwell, Mona I; Brown, Karen; Gaskell, Margaret; Wright, Elizabeth; Poirier, Miriam C

2005-03-01

Liver homogenates from rats fed tamoxifen (TAM) in the diet were shared among four different laboratories. TAM-DNA adducts were assayed by high pressure liquid chromatography-electrospray tandem mass spectrometry (HPLC-ES-MS/MS), TAM-DNA chemiluminescence immunoassay (TAM-DNA CIA), and (32)P-postlabeling with either thin layer ((32)P-P-TLC) or liquid chromatography ((32)P-P-HPLC) separation. In the first study, rats were fed a diet containing 500 p.p.m. TAM for 2 months, and the values for measurements of the (E)-alpha-(deoxyguanosin-N(2)-yl)-tamoxifen (dG-N(2)-TAM) adduct in replicate rat livers varied by 3.5-fold when quantified using 'in house' TAM-DNA standards, or other approaches where appropriate. In the second study, rats were fed 0, 50, 250 or 500 p.p.m. TAM for 2 months, and TAM-DNA values were quantified using both 'in house' approaches as well as a newly synthesized [N-methyl-(3)H]TAM-DNA standard that was shared among all the participating groups. In the second study, the total TAM-DNA adduct values varied by 2-fold, while values for the dG-N(2)-TAM varied by 2.5-fold. Ratios of dG-N(2)-TAM:(E)-alpha-(deoxyguanosin-N(2)-yl)-N-desmethyltamoxifen (dG-N(2)-N-desmethyl-TAM) in the second study were approximately 1:1 over the range of doses examined. The study demonstrated a remarkably good agreement for TAM-DNA adduct measurements among the diverse methods employed.

PGC-1alpha increases skeletal muscle lactate uptake by increasing the expression of MCT1 but not MCT2 or MCT4.

PubMed

Benton, Carley R; Yoshida, Yuko; Lally, James; Han, Xiao-Xia; Hatta, Hideo; Bonen, Arend

2008-09-17

We examined the relationship between PGC-1alpha protein; the monocarboxylate transporters MCT1, 2, and 4; and CD147 1) among six metabolically heterogeneous rat muscles, 2) in chronically stimulated red (RTA) and white tibialis (WTA) muscles (7 days), and 3) in RTA and WTA muscles transfected with PGC-1alpha-pcDNA plasmid in vivo. Among rat hindlimb muscles, there was a strong positive association between PGC-1alpha and MCT1 and CD147, and between MCT1 and CD147. A negative association was found between PGC-1alpha and MCT4, and CD147 and MCT4, while there was no relationship between PGC-1alpha or CD147 and MCT2. Transfecting PGC-1alpha-pcDNA plasmid into muscle increased PGC-1alpha protein (RTA +23%; WTA +25%) and induced the expression of MCT1 (RTA +16%; WTA +28%), but not MCT2 and MCT4. As a result of the PGC-1alpha-induced upregulation of MCT1 and its chaperone CD147 (+29%), there was a concomitant increase in the rate of lactate uptake (+20%). In chronically stimulated muscles, the following proteins were upregulated, PGC-1alpha in RTA (+26%) and WTA (+86%), MCT1 in RTA (+61%) and WTA (+180%), and CD147 in WTA (+106%). In contrast, MCT4 protein expression was not altered in either RTA or WTA muscles, while MCT2 protein expression was reduced in both RTA (-14%) and WTA (-10%). In these studies, whether comparing oxidative capacities among muscles or increasing their oxidative capacities by PGC-1alpha transfection and chronic muscle stimulation, there was a strong relationship between the expression of PGC-1alpha and MCT1, and PGC-1alpha and CD147 proteins. Thus, MCT1 and CD147 belong to the family of metabolic genes whose expression is regulated by PGC-1alpha in skeletal muscle.

The organization and evolution of the Responder satellite in species of the Drosophila melanogaster group: dynamic evolution of a target of meiotic drive.

PubMed

Larracuente, Amanda M

2014-11-25

Satellite DNA can make up a substantial fraction of eukaryotic genomes and has roles in genome structure and chromosome segregation. The rapid evolution of satellite DNA can contribute to genomic instability and genetic incompatibilities between species. Despite its ubiquity and its contribution to genome evolution, we currently know little about the dynamics of satellite DNA evolution. The Responder (Rsp) satellite DNA family is found in the pericentric heterochromatin of chromosome 2 of Drosophila melanogaster. Rsp is well-known for being the target of Segregation Distorter (SD)- an autosomal meiotic drive system in D. melanogaster. I present an evolutionary genetic analysis of the Rsp family of repeats in D. melanogaster and its closely-related species in the melanogaster group (D. simulans, D. sechellia, D. mauritiana, D. erecta, and D. yakuba) using a combination of available BAC sequences, whole genome shotgun Sanger reads, Illumina short read deep sequencing, and fluorescence in situ hybridization. I show that Rsp repeats have euchromatic locations throughout the D. melanogaster genome, that Rsp arrays show evidence for concerted evolution, and that Rsp repeats exist outside of D. melanogaster, in the melanogaster group. The repeats in these species are considerably diverged at the sequence level compared to D. melanogaster, and have a strikingly different genomic distribution, even between closely-related sister taxa. The genomic organization of the Rsp repeat in the D. melanogaster genome is complex-it exists of large blocks of tandem repeats in the heterochromatin and small blocks of tandem repeats in the euchromatin. My discovery of heterochromatic Rsp-like sequences outside of D. melanogaster suggests that SD evolved after its target satellite and that the evolution of the Rsp satellite family is highly dynamic over a short evolutionary time scale (<240,000 years).

Behavior of a cloned murine interferon alpha/beta receptor expressed in homospecific or heterospecific background.

PubMed Central

Uzé, G; Lutfalla, G; Bandu, M T; Proudhon, D; Mogensen, K E

1992-01-01

A murine interferon (IFN) alpha/beta receptor was cloned from the IFN-sensitive L1210 cell line on the basis of its homology with the human receptor. A combination of methods that includes the screening of random-primed and oligo(dT)-primed cDNA libraries and polymerase chain reactions with a single-side specificity was used. At the amino acid level, the murine IFN-alpha/beta shows 46% identity with its human counterpart. Both human WISH cells presenting a low sensitivity to mouse IFN and a murine L1210 mutant subline that does not express the receptor have been stably transfected with the murine IFN-alpha/beta receptor. Whereas transfected human cells became sensitive to a limited number of mouse IFN-alpha/beta subtypes, the transfected murine L1210 mutant was found to be fully complemented and became sensitive to all mouse IFN-alpha/beta subtypes tested, including those that were not active on transfected human cells. These results strongly suggest that the receptor described here is implicated in the mediation of the activities of all murine IFN-alpha/beta subtypes. Images PMID:1533935

Spacing requirements for interactions between the C-terminal domain of the alpha subunit of Escherichia coli RNA polymerase and the cAMP receptor protein.

PubMed Central

Lloyd, G S; Busby, S J; Savery, N J

1998-01-01

During transcription initiation at bacterial promoters, the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD) can interact with DNA-sequence elements (known as UP elements) and with activator proteins. We have constructed a series of semi-synthetic promoters carrying both an UP element and a consensus DNA-binding site for the Escherichia coli cAMP receptor protein (CRP; a factor that activates transcription by making direct contacts with alphaCTD). At these promoters, the UP element was located at a variety of distances upstream of the CRP-binding site, which was fixed at position -41.5 bp upstream of the transcript start. At some positions, the UP element caused enhanced promoter activity whereas, at other positions, it had very little effect. In no case was the CRP-dependence of the promoter relieved. DNase I and hydroxyl-radical footprinting were used to study ternary RNA polymerase-CRP-promoter complexes formed at two of the most active of these promoters, and co-operativity between the binding of CRP and purified alpha subunits was studied. The footprints show that alphaCTD binds to the UP element as it is displaced upstream but that this displacement does not prevent alphaCTD from being contacted by CRP. Models to account for this are discussed. PMID:9461538

A facile and efficient method to modify gold nanorods with thiolated DNA at a low pH value.

PubMed

Shi, Dangwei; Song, Chen; Jiang, Qiao; Wang, Zhen-Gang; Ding, Baoquan

2013-03-28

We report a simple, rapid and efficient strategy for modification of gold nanorods (AuNRs) with thiolated DNA at low solution pH and high salt concentration. DNA functionalized AuNRs were then used to assemble with DNA modified gold nanoparticles to form discrete satellite nanostructures.

Ultraviolet observations of cool stars. IV - Intensities of Lyman-alpha and Mg II in epsilon Pegasi and epsilon Eridani, and line width-luminosity correlations

NASA Technical Reports Server (NTRS)

Mcclintock, W.; Linsky, J. L.; Henry, R. C.; Moos, H. W.

1975-01-01

A spectrometer on the Copernicus satellite has been used to confirm the existence of a line width-luminosity relation for the Ly-alpha and Mg II 2800-A chromospheric emission lines in K-type stars by observation of a K2 dwarf (epsilon Eri) and a K2 supergiant (epsilon Peg). Combined with previously reported observations of lines in three K giants (alpha Boo, alpha Tau, and beta Gem), the data are consistent with an identical dependence of line width on absolute visual magnitude for the Ca II K, Ly-alpha, and Mg II 2795-A lines. Surface fluxes of Ly-alpha, Mg II 2800-A, and O V 1218-A (upper limit) for epsilon Eri, and of Mg II 2800-A for epsilon Peg are also compared with values reported previously for the three giant stars.

Biophysical and transfection studies of the diC(14)-amidine/DNA complex.

PubMed Central

Cherezov, Vadim; Qiu, Hong; Pector, Veronique; Vandenbranden, Michel; Ruysschaert, Jean-Marie; Caffrey, Martin

2002-01-01

Liposomes of the synthetic cationic lipid, N-t-butyl-N'-tetradecylamino-propionamidine (diC(14)-amidine), efficiently ports DNA into mammalian cells in the absence of other (neutral) lipids. The compositional simplicity of this transfection mix makes it attractive from a formulation perspective. We have used low- and wide-angle x-ray diffraction and polarized light microscopy to characterize the thermotropic phase behavior and microstructure of diC(14)-amidine and of the lipid/DNA (circular plasmid, 5.4 kb) complex with a view to understanding the structure of the complex and its role in transfection. Upon heating, the lipid in buffer undergoes a lamellar crystalline (L(c), d(001) = 41.7 A)-to-lamellar liquid crystal (L(c)(alpha), d(001) depends on hydration and T) transition at approximately 40 degrees C. Sonicated lipid vesicles with a reported transition temperature of approximately 23 degrees C complex with DNA. Complex formation is complete at a DNA/lipid mole ratio (rho) of 0.8. Adding DNA to the lipid causes d(001) of the multilayered complex to drop from 52 to 49 A as rho rises from 0.03 to 1.64. The minimal DNA-DNA duplex separation observed is 26 A, consistent with the close packing of B-DNA. Lipid bilayers in the complex undergo a lamellar gel (L(c)(beta))-to-L(c)(alpha) (superscript c refers to complex) transition at approximately 23 degrees C. Transfection efficiency was maximized at rho = 0.4. The structure and transfection data combined suggest that densely packaged DNA in a net positively charged complex is essential for transfection. PMID:12023234

Evolutionary dynamics and sites of illegitimate recombination revealed in the interspersion and sequence junctions of two nonhomologous satellite DNAs in cactophilic Drosophila species.

PubMed

Kuhn, G C S; Teo, C H; Schwarzacher, T; Heslop-Harrison, J S

2009-05-01

Satellite DNA (satDNA) is a major component of genomes but relatively little is known about the fine-scale organization of unrelated satDNAs residing at the same chromosome location, and the sequence structure and dynamics of satDNA junctions. We studied the organization and sequence junctions of two nonhomologous satDNAs, pBuM and DBC-150, in three species from the neotropical Drosophila buzzatii cluster (repleta group). In situ hybridization to microchromosomes, interphase nuclei and extended DNA fibers showed frequent interspersion of the two satellites in D. gouveai, D. antonietae and, to a lesser extent, D. seriema. We isolated by PCR six pBuM x DBC-150 junctions: four are exclusive to D. gouveai and two are exclusive to D. antonietae. The six junction breakpoints occur at different positions within monomers, suggesting independent origin. Four junctions showed abrupt transitions between the two satellites, whereas two junctions showed a distinct 10 bp tandem duplication before the junction. Unlike pBuM, DBC-150 junction repeats are more variable than randomly cloned monomers and showed diagnostic features in common to a 3-monomer higher-order repeat seen in the sister species D. serido. The high levels of interspersion between pBuM and DBC-150 repeats suggest extensive rearrangements between the two satellites, maybe favored by specific features of the microchromosomes. Our interpretation is that the junctions evolved by multiples events of illegitimate recombination between nonhomologous satDNA repeats, with subsequent rounds of unequal crossing-over expanding the copy number of some of the junctions.

The Delta Low-Inclination Satellite Concept, an Opportunity to Enhance the Science Return of the Swarm Mission

NASA Astrophysics Data System (ADS)

Hulot, G.; Leger, J. M.; Olsen, N.; Stolle, C.; Chulliat, A.; Kuvshinov, A. V.; Vigneron, P.; Lesur, V.; Shimizu, H.; Sreenivasan, B.

2015-12-01

ESA's Swarm mission aims at studying all sources of Earth's magnetic field. It consists of two satellites (Alpha and Charlie), which fly side-by-side on near polar orbits at an altitude of slightly less than 500 km, and of a third satellite (Bravo) on a similar but slightly more polar and higher orbit, which progressively drifts with respect to that of Alpha and Charlie. This orbital configuration has proven extremely valuable, as evidenced by the many results already obtained from the first two years of the mission. These results, however, also reveal that geomagnetic field modeling and investigation efforts are now hampered by the still limited local time coverage provided by this constellation. This affects our ability to accurately characterize time changes in the ionospheric and magnetospheric field contributions, and to model the electrical conductivity of the Earth's mantle. It also indirectly limits our ability to model the core and lithospheric field. More generally, many of the "residual signals" detected in the very accurate magnetic data of the Swarm mission can still not fully be exploited. Further increasing the scientific return of the Swarm mission by squeezing more out of these data, however, would be possible if a fourth "Delta" satellite were to be launched soon enough to join the constellation at a similar altitude but much lower inclination orbit (such as 60°). Such a satellite would provide less geographical coverage but a much faster mapping of all local times over these latitudes. In this presentation we will present the rational for such a Delta mission and discuss the benefit it would bring, as well as plans currently under consideration to build a small satellite that could meet the corresponding requirements.

Cloning, sequencing, and expression of the gene coding for bile acid 7 alpha-hydroxysteroid dehydrogenase from Eubacterium sp. strain VPI 12708.

PubMed Central

Baron, S F; Franklund, C V; Hylemon, P B

1991-01-01

Southern blot analysis indicated that the gene encoding the constitutive, NADP-linked bile acid 7 alpha-hydroxysteroid dehydrogenase of Eubacterium sp. strain VPI 12708 was located on a 6.5-kb EcoRI fragment of the chromosomal DNA. This fragment was cloned into bacteriophage lambda gt11, and a 2.9-kb piece of this insert was subcloned into pUC19, yielding the recombinant plasmid pBH51. DNA sequence analysis of the 7 alpha-hydroxysteroid dehydrogenase gene in pBH51 revealed a 798-bp open reading frame, coding for a protein with a calculated molecular weight of 28,500. A putative promoter sequence and ribosome binding site were identified. The 7 alpha-hydroxysteroid dehydrogenase mRNA transcript in Eubacterium sp. strain VPI 12708 was about 0.94 kb in length, suggesting that it is monocistronic. An Escherichia coli DH5 alpha transformant harboring pBH51 had approximately 30-fold greater levels of 7 alpha-hydroxysteroid dehydrogenase mRNA, immunoreactive protein, and specific activity than Eubacterium sp. strain VPI 12708. The 7 alpha-hydroxysteroid dehydrogenase purified from the pBH51 transformant was similar in subunit molecular weight, specific activity, and kinetic properties to that from Eubacterium sp. strain VPI 12708, and it reached with antiserum raised against the authentic enzyme on Western immunoblots. Alignment of the amino acid sequence of the 7 alpha-hydroxysteroid dehydrogenase with those of 10 other pyridine nucleotide-linked alcohol/polyol dehydrogenases revealed six conserved amino acid residues in the N-terminal regions thought to function in coenzyme binding. Images PMID:1856160

Analysis of Claviceps africana and C. sorghi from India using AFLPs, EF-1alpha gene intron 4, and beta-tubulin gene intron 3.

PubMed

Tooley, Paul W; Bandyopadhyay, Ranajit; Carras, Marie M; Pazoutová, Sylvie

2006-04-01

Isolates of Claviceps causing ergot on sorghum in India were analysed by AFLP analysis, and by analysis of DNA sequences of the EF-1alpha gene intron 4 and beta-tubulin gene intron 3 region. Of 89 isolates assayed from six states in India, four were determined to be C. sorghi, and the rest C. africana. A relatively low level of genetic diversity was observed within the Indian C. africana population. No evidence of genetic exchange between C. africana and C. sorghi was observed in either AFLP or DNA sequence analysis. Phylogenetic analysis was conducted using DNA sequences from 14 different Claviceps species. A multigene phylogeny based on the EF-1alpha gene intron 4, the beta-tubulin gene intron 3 region, and rDNA showed that C. sorghi grouped most closely with C. gigantea and C. africana. Although the Claviceps species we analysed were closely related, they colonize hosts that are taxonomically very distinct suggesting that there is no direct coevolution of Claviceps with its hosts.

Structural Determinants of DNA Binding by a P. falciparum ApiAP2 Transcriptional Regulator

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lindner, Scott E.; De Silva, Erandi K.; Keck, James L.

2010-11-05

Putative transcription factors have only recently been identified in the Plasmodium spp., with the major family of regulators comprising the Apicomplexan Apetala2 (AP2) proteins. To better understand the DNA-binding mechanisms of these transcriptional regulators, we characterized the structure and in vitro function of an AP2 DNA-binding domain from a prototypical Apicomplexan AP2 protein, PF14{_}0633 from Plasmodium falciparum. The X-ray crystal structure of the PF14{_}0633 AP2 domain bound to DNA reveals a {beta}-sheet fold that binds the DNA major groove through base-specific and backbone contacts; a prominent {alpha}-helix supports the {beta}-sheet structure. Substitution of predicted DNA-binding residues with alanine weakened ormore » eliminated DNA binding in solution. In contrast to plant AP2 domains, the PF14{_}0633 AP2 domain dimerizes upon binding to DNA through a domain-swapping mechanism in which the {alpha}-helices of the AP2 domains pack against the {beta}-sheets of the dimer mates. DNA-induced dimerization of PF14{_}0633 may be important for tethering two distal DNA loci together in the nucleus and/or for inducing functional rearrangements of its domains to facilitate transcriptional regulation. Consistent with a multisite binding mode, at least two copies of the consensus sequence recognized by PF14{_}0633 are present upstream of a previously identified group of sporozoite-stage genes. Taken together, these findings illustrate how Plasmodium has adapted the AP2 DNA-binding domain for genome-wide transcriptional regulation.« less

Induction of experimental bone metastasis in mice by transfection of integrin alpha 4 beta 1 into tumor cells.

PubMed Central

Matsuura, N.; Puzon-McLaughlin, W.; Irie, A.; Morikawa, Y.; Kakudo, K.; Takada, Y.

1996-01-01

Cell adhesion receptors (eg, integrins and CD44) play an important role in invasion and metastasis during tumor progression. The increase in integrin alpha 4 beta 1 expression on primary melanomas has been reported to significantly correlate with the development of metastases. alpha 4 beta 1 is a cell surface heterodimer that mediates cell-cell and cell-extracellular matrix interactions through adhesion to vascular cell adhesion molecule (VCAM)-1 and to the IIICS region of fibronectin. To test the effects of alpha 4 beta 1 expression on tumor cell metastasis, Chinese hamster ovary cells were transfected with human alpha 4 cDNA. Whereas alpha 4-negative Chinese hamster ovary cells developed only pulmonary metastasis, alpha 4-positive Chinese hamster ovary cells developed bone and pulmonary metastasis in 3 to 4 weeks when injected intravenously into nude mice. Bone metastasis was inhibited by antibody against alpha 4 or VCAM-1. Expression of alpha 3 beta 1, alpha 6 beta 1, or alpha V beta 1 did not induce bone metastasis. Expression of alpha 4 beta 1 also induced bone metastasis in K562 human erythroleukemia cells injected into SCID mice. These results demonstrate that alpha 4 beta 1 can induce tumor cell trafficking to bone, probably via interaction with VCAM-1 that is constitutively expressed on bone marrow stromal cells. Images Figure 1 Figure 3 PMID:8546226

Electronic Characterization of Au/DNA/ITO Metal-Semiconductor-Metal Diode and Its Application as a Radiation Sensor.

PubMed

Al-Ta'ii, Hassan Maktuff Jaber; Periasamy, Vengadesh; Amin, Yusoff Mohd

2016-01-01

Deoxyribonucleic acid or DNA molecules expressed as double-stranded (DSS) negatively charged polymer plays a significant role in electronic states of metal/silicon semiconductor structures. Electrical parameters of an Au/DNA/ITO device prepared using self-assembly method was studied by using current-voltage (I-V) characteristic measurements under alpha bombardment at room temperature. The results were analyzed using conventional thermionic emission model, Cheung and Cheung's method and Norde's technique to estimate the barrier height, ideality factor, series resistance and Richardson constant of the Au/DNA/ITO structure. Besides demonstrating a strongly rectifying (diode) characteristic, it was also observed that orderly fluctuations occur in various electrical parameters of the Schottky structure. Increasing alpha radiation effectively influences the series resistance, while the barrier height, ideality factor and interface state density parameters respond linearly. Barrier height determined from I-V measurements were calculated at 0.7284 eV for non-radiated, increasing to about 0.7883 eV in 0.036 Gy showing an increase for all doses. We also demonstrate the hypersensitivity phenomena effect by studying the relationship between the series resistance for the three methods, the ideality factor and low-dose radiation. Based on the results, sensitive alpha particle detectors can be realized using Au/DNA/ITO Schottky junction sensor.

Electronic Characterization of Au/DNA/ITO Metal-Semiconductor-Metal Diode and Its Application as a Radiation Sensor

PubMed Central

Al-Ta’ii, Hassan Maktuff Jaber; Periasamy, Vengadesh; Amin, Yusoff Mohd

2016-01-01

Deoxyribonucleic acid or DNA molecules expressed as double-stranded (DSS) negatively charged polymer plays a significant role in electronic states of metal/silicon semiconductor structures. Electrical parameters of an Au/DNA/ITO device prepared using self-assembly method was studied by using current–voltage (I-V) characteristic measurements under alpha bombardment at room temperature. The results were analyzed using conventional thermionic emission model, Cheung and Cheung’s method and Norde’s technique to estimate the barrier height, ideality factor, series resistance and Richardson constant of the Au/DNA/ITO structure. Besides demonstrating a strongly rectifying (diode) characteristic, it was also observed that orderly fluctuations occur in various electrical parameters of the Schottky structure. Increasing alpha radiation effectively influences the series resistance, while the barrier height, ideality factor and interface state density parameters respond linearly. Barrier height determined from I–V measurements were calculated at 0.7284 eV for non-radiated, increasing to about 0.7883 eV in 0.036 Gy showing an increase for all doses. We also demonstrate the hypersensitivity phenomena effect by studying the relationship between the series resistance for the three methods, the ideality factor and low-dose radiation. Based on the results, sensitive alpha particle detectors can be realized using Au/DNA/ITO Schottky junction sensor. PMID:26799703

Feasibility of hydrogen density estimation from tomographic sensing of Lyman alpha emission

NASA Astrophysics Data System (ADS)

Waldrop, L.; Kamalabadi, F.; Ren, D.

2015-12-01

In this work, we describe the scientific motivation, basic principles, and feasibility of a new approach to the estimation of neutral hydrogen (H) density in the terrestrial exosphere based on the 3-D tomographic sensing of optically thin H emission at 121.6 nm (Lyman alpha). In contrast to existing techniques, Lyman alpha tomography allows for model-independent reconstruction of the underlying H distribution in support of investigations regarding the origin and time-dependent evolution of exospheric structure. We quantitatively describe the trade-off space between the measurement sampling rate, viewing geometry, and the spatial and temporal resolution of the reconstruction that is supported by the data. We demonstrate that this approach is feasible from either earth-orbiting satellites such as the stereoscopic NASA TWINS mission or from a CubeSat platform along a trans-exosphere trajectory such as that enabled by the upcoming Exploration Mission 1 launch.

Crystallisation of alpha-crustacyanin, the lobster carapace astaxanthin-protein: results from EURECA

NASA Astrophysics Data System (ADS)

Zagalsky, P. F.; Wright, C. E.; Parsons, M.

1995-08-01

Crystallisation of alpha-crustacyanin, the lobster carapace astaxanthin-protein was attempted under microgravity conditions in EURECA satellite using liquid-liquid diffusion with polyethyleneglycol (PEG) as precipitant; in a second reaction chamber phenol and dioxan were used as additives to prevent composite crystal growth. Crystals of alpha-crustacyanin grown under microgravity from PEG were larger than those grown terrestrially in the same apparatus under otherwise identical conditions. On retrieval, the crystals from PEG were shown to be composite and gave a powder diffraction pattern. The second reaction chamber showed leakage on retrieval and had also been subjected to rapid temperature variation during flight. Crystal fragments were nevertheless recovered but showed a powder diffraction pattern. It is concluded, certainly for liquid-liquid diffusion using PEG alone, that, for crustacyanin, although microgravity conditions resulted in an increase in dimensions of crystals, a measurable improvement in molecular ordering was not achieved.

Anti-inflammatory effect of resveratrol on TNF-{alpha}-induced MCP-1 expression in adipocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhu Jian; Key Laboratory of Human Functional Genomics of Jiangsu Province, School of Basic Medical Science, Jiangsu Province Diabetes Center, Nanjing Medical University, 140 Hanzhong Road, Nanjing 210029; Yong Wei

2008-05-02

Chronic low-grade inflammation characterized by adipose tissue macrophage accumulation and abnormal cytokine production is a key feature of obesity and type 2 diabetes. Adipose-tissue-derived monocyte chemoattractant protein (MCP)-1, induced by cytokines, has been shown to play an essential role in the early events during macrophage infiltration into adipose tissue. In this study we investigated the effects of resveratrol upon both tumor necrosis factor (TNF)-{alpha}-induced MCP-1 gene expression and its underlying signaling pathways in 3T3-L1 adipoctyes. Resveratrol was found to inhibit TNF-{alpha}-induced MCP-1 secretion and gene transcription, as well as promoter activity, which based on down-regulation of TNF-{alpha}-induced MCP-1 transcription. Nuclearmore » factor (NF)-{kappa}B was determined to play a major role in the TNF-{alpha}-induced MCP-1 expression. Further analysis showed that resveratrol inhibited DNA binding activity of the NF-{kappa}B complex and subsequently suppressed NF-{kappa}B transcriptional activity in TNF-{alpha}-stimulated cells. Finally, the inhibition of MCP-1 may represent a novel mechanism of resveratrol in preventing obesity-related pathologies.« less

Inhibition of gamma-radiation induced DNA damage in plasmid pBR322 by TMG, a water-soluble derivative of vitamin E.

PubMed

Rajagopalan, Rema; Wani, Khalida; Huilgol, Nagaraj G; Kagiya, Tsutomu V; Nair, Cherupally K Krishnan

2002-06-01

Alpha-tocopherol monoglucoside (TMG), a water-soluble derivative of alpha-tocopherol, has been examined for its ability to protect DNA against radiation-induced strand breaks. Gamma radiation, up to a dose of 6 Gy (dose rate, 0.7 Gy/minute), induced a dose-dependent increase in single strand breaks (SSBs) in plasmid pBR322 DNA. TMG inhibited the formation of gamma-radiation induced DNA single strand breaks (SSBs) in a concentration-dependent manner; 500 microM of TMG protected the single strand breaks completely. It also protected thymine glycol formation induced by gamma-radiation in a dose-dependent manner, based on an estimation of thymine glycol by HPLC.

DNA methylation of retrotransposons, DNA transposons and genes in sugar beet (Beta vulgaris L.).

PubMed

Zakrzewski, Falk; Schmidt, Martin; Van Lijsebettens, Mieke; Schmidt, Thomas

2017-06-01

The methylation of cytosines shapes the epigenetic landscape of plant genomes, coordinates transgenerational epigenetic inheritance, represses the activity of transposable elements (TEs), affects gene expression and, hence, can influence the phenotype. Sugar beet (Beta vulgaris ssp. vulgaris), an important crop that accounts for 30% of worldwide sugar needs, has a relatively small genome size (758 Mbp) consisting of approximately 485 Mbp repetitive DNA (64%), in particular satellite DNA, retrotransposons and DNA transposons. Genome-wide cytosine methylation in the sugar beet genome was studied in leaves and leaf-derived callus with a focus on repetitive sequences, including retrotransposons and DNA transposons, the major groups of repetitive DNA sequences, and compared with gene methylation. Genes showed a specific methylation pattern for CG, CHG (H = A, C, and T) and CHH sites, whereas the TE pattern differed, depending on the TE class (class 1, retrotransposons and class 2, DNA transposons). Along genes and TEs, CG and CHG methylation was higher than that of adjacent genomic regions. In contrast to the relatively low CHH methylation in retrotransposons and genes, the level of CHH methylation in DNA transposons was strongly increased, pointing to a functional role of asymmetric methylation in DNA transposon silencing. Comparison of genome-wide DNA methylation between sugar beet leaves and callus revealed a differential methylation upon tissue culture. Potential epialleles were hypomethylated (lower methylation) at CG and CHG sites in retrotransposons and genes and hypermethylated (higher methylation) at CHH sites in DNA transposons of callus when compared with leaves. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

Organocatalyzed asymmetric alpha-oxidation, alpha-aminoxylation and alpha-amination of carbonyl compounds.

PubMed

Vilaivan, Tirayut; Bhanthumnavin, Worawan

2010-02-11

Organocatalytic asymmetric alpha-oxidation and amination reactions of carbonyl compounds are highly useful synthetic methodologies, especially in generating chiral building blocks that previously have not been easily accessible by traditional methods. The concept is relatively new and therefore the list of new catalysts, oxidizing and aminating reagents, as well as new substrates, are expanding at an amazing rate. The scope of this review includes new reactions and catalysts, mechanistic aspects and synthetic applications of alpha-oxidation, hydroxylation, aminoxylation, amination, hydrazination, hydroxyamination and related alpha-heteroatom functionalization of aldehydes, ketones and related active methylene compounds published during 2005-2009.

Bile alcohol metabolism in man. Conversion of 5beta-cholestane-3alpha, 7alpha,12alpha, 25-tetrol to cholic acid.

PubMed Central

Salen, G; Shefer, S; Setoguchi, T; Mosbach, E H

1975-01-01

To study the role of C25-HYDROXY BILE ALCOHOLS AS PRECURSORS OF CHOlic acid, [G-3-H]5beta-cholestane-3alpha,7alpha12alpha,25-tetrol was administered intravenously to two subjects with cerebrotendinous xanthomatosis (CTX) and two normal individuals. One day after pulse labeling, radioactivity was present in the cholic acid isolated from the bile and feces of the subjects with CTX and the bile of the normal individuals. In the two normal subjects, the sp act decay curves of [G-3-H]-cholic acid were exponential, and no traces of [G-3-H]-5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol were detected. In contrast, appreciable quantities of labeled 5beta-cholestane-3alpha,-7aopha,12alpha,25-tetrol were present in the bile and feces of the CTX subjects. The sp act vs. time curves of fecal [G-3-H]5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol and [G-3-H]-cholic acid showed a precursor-product relationship. Although these results suggest that 5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol may be a precursor of cholic acid in man, the possibility that C26-hydroxy intermediates represent the normal pathway can not be excluded. PMID:1141434

Effect of long-term exposure to mobile phone radiation on alpha-Int1 gene sequence of Candida albicans

PubMed Central

Shahin-jafari, Ariyo; Bayat, Mansour; Shahhosseiny, Mohammad Hassan; Tajik, Parviz; Roudbar-mohammadi, Shahla

2015-01-01

Over the last decade, communication industries have witnessed a tremendous expansion, while, the biological effects of electromagnetic waves have not been fully elucidated. Current study aimed at evaluating the mutagenic effect of long-term exposure to 900-MHz radiation on alpha-Int1 gene sequences of Candida albicans. A standard 900 MHz radiation generator was used for radiation. 10 ml volumes from a stock suspension of C. albicans were transferred into 10 polystyrene tubes. Five tubes were exposed at 4 °C to a fixed magnitude of radiation with different time periods of 10, 70, 210, 350 and 490 h. The other 5 tubes were kept far enough from radiation. The samples underwent genomic DNA extraction. PCR amplification of alpha-Int1 gene sequence was done using one set of primers. PCR products were resolved using agarose gel electrophoresis and the nucleotide sequences were determined. All samples showed a clear electrophoretic band around 441 bp and further sequencing revealed the amplified DNA segments are related to alpha-Int1 gene of the yeast. No mutations in the gene were seen in radiation exposed samples. Long-term exposure of the yeast to mobile phone radiation under the above mentioned conditions had no mutagenic effect on alpha-Int1 gene sequence. PMID:27081370

Oestrogen receptor-alpha activation augments post-exercise myoblast proliferation.

PubMed

Thomas, A; Bunyan, K; Tiidus, P M

2010-01-01

Our laboratory has shown that oestrogen acts to augment myoblast (satellite cell) activation, proliferation and total number and that this may occur through an oestrogen receptor (OR)-mediated mechanism. The purpose of this study was to further investigate the mechanism of oestrogen influence on augmentation of post-exercise myoblast numbers through use of a specific OR-alpha agonist, propyl pyrazole triol (PPT). Ovariectomized rats were used (n = 64) and separated into four groups: sham, oestrogen supplemented, agonist supplemented, and a combined oestrogen and agonist supplemented group. These groups were further subdivided into control (unexercised) and exercise groups. Surgical removal of white vastus and soleus muscles was performed 72 h post-exercise. Muscle samples were immunostained for the myoblast markers Pax7 and MyoD. A significant increase in total (Pax7-positive) and activated (MyoD-positive) myoblasts was found in all groups post-exercise. A further significant augmentation of total and activated myoblasts occurred in oestrogen supplemented, agonist supplemented and the combined oestrogen and agonist supplemented groups post-exercise in white vastus and soleus muscles relative to unsupplemented animals. These results demonstrate that both oestrogen and the specific OR-alpha receptor agonist, PPT, can significantly and to similar degrees augment myoblast number and activation following exercise-induced muscle damage. This suggests that oestrogen acts through an OR-mediated mechanism to stimulate myoblast proliferation following exercise, with OR-alpha playing a primary role.

Contribution of alpha3(IV)alpha4(IV)alpha5(IV) Collagen IV to the Mechanical Properties of the Glomerular Basement Membrane

NASA Astrophysics Data System (ADS)

Gyoneva, Lazarina

The glomerular basement membrane (GBM) is a vital part of the blood-urine filtration barrier in the kidneys. In healthy GBMs, the main tension-resisting component is alpha3(IV)alpha4(IV)alpha5(IV) type IV collagen, but in some diseases it is replaced by other collagen IV isoforms. As a result, the GBM becomes leaky and disorganized, ultimately resulting in kidney failure. Our goal is to understanding the biomechanical aspects of the alpha3(IV)alpha4(IV)alpha5(IV) chains and how their absence could be responsible for (1) the initial injury to the GBM and (2) progression to kidney failure. A combination of experiments and computational models were designed for that purpose. A model basement membrane was used to compare experimentally the distensibility of tissues with the alpha3(IV)alpha4(IV)alpha5(IV) chains present and missing. The experiments showed basement membranes containing alpha3(IV)alpha4(IV)alpha5(IV) chains were less distensible. It has been postulated that the higher level of lateral cross-linking (supercoiling) in the alpha3(IV)alpha4(IV)alpha5(IV) networks contributes additional strength/stability to basement membranes. In a computational model of supercoiled networks, we found that supercoiling greatly increased the stiffness of collagen IV networks but only minimally decreased the permeability, which is well suited for the needs of the GBM. It is also known that the alpha3(IV)alpha4(IV)alpha5(IV) networks are more protected from enzymatic degradation, and we explored their significance in GBM remodeling. Our simulations showed that the more protected network was needed to prevent the system from entering a dangerous feedback cycle due to autoregulation mechanisms in the kidneys. Overall, the work adds to the evidence of biomechanical differences between the alpha3(IV)alpha4(IV)alpha5(IV) networks and other collagen IV networks, points to supercoiling as the main source of biomechanical differences, discusses the suitability of alpha3(IV)alpha4(IV)alpha

THE FORM AND STRUCTURE OF KINETOPLAST DNA OF CRITHIDIA

PubMed Central

Renger, Hartmut C.; Wolstenholme, David R.

1972-01-01

Cesium chloride centrifugation of each of the DNAs extracted from eight strains of Crithidia revealed a main band at ρ = 1.717 g/cm3 and a satellite band varying from ρ = 1.701 to 1.705 g/cm3 for the different strains By electron microscopy each DNA was shown to include circular molecules, 0.69–0.80 µ in mean contour length, and large, topologically two-dimensional masses of DNA in which the molecules appeared in the form of rosettes. DNA isolated from kinetoplast fractions of Crithidia acanthocephali was shown to consist of light satellite DNA and to be mainly in the form of large masses, 0.8 µ (mol wt = 1.54 x 106 daltons) circular molecules, and a few long, linear molecules. The results of experiments involving ultracentrifugation, heating, and quenching, sonication, and endodeoxyribonuclease digestion, combined with electron microscopy, are consistent with the following hypothesis. The large DNA masses are associations of 0.8 µ circles which are mainly covalently closed. The circles are held together in groups (the rosettes) of up to 46 by the topological interlocking of each circle with many other circles in the group. A group of circles is attached to an adjacent group by one or more circles, each interlocking with many circles of both groups. Each of the associations comprises, on the average, about 27,000 circles (total mol wt ≃ 41 x 109 daltons). A model is proposed for the in situ arrangement of the associations which takes into consideration their form and structure, and appearance in thin sections PMID:5040863

Increased virulence and competitive advantage of a/alpha over a/a or alpha/alpha offspring conserves the mating system of Candida albicans.

PubMed

Lockhart, Shawn R; Wu, Wei; Radke, Joshua B; Zhao, Rui; Soll, David R

2005-04-01

The majority of Candida albicans strains in nature are a/alpha and must undergo homozygosis to a/a or alpha/alpha to mate. Here we have used a mouse model for systemic infection to test the hypothesis that a/alpha strains predominate in nature because they have a competitive advantage over a/a and alpha/alpha offspring in colonizing hosts. Single-strain injection experiments revealed that a/alpha strains were far more virulent than either their a/a or alpha/alpha offspring. When equal numbers of parent a/alpha and offspring a/a or alpha/alpha cells were co-injected, a/alpha always exhibited a competitive advantage at the time of extreme host morbidity or death. When equal numbers of an engineered a/a/alpha2 strain and its isogenic a/a parent strain were co-injected, the a/a/alpha2 strain exhibited a competitive advantage at the time of host morbidity or death, suggesting that the genotype of the mating-type (MTL) locus, not associated genes on chromosome 5, provides a competitive advantage. We therefore propose that heterozygosity at the MTL locus not only represses white-opaque switching and genes involved in the mating process, but also affects virulence, providing a competitive advantage to the a/alpha genotype that conserves the mating system of C. albicans in nature.

Inhibition of herpes simplex virus DNA polymerase by purine ribonucleoside monophosphates.

PubMed

Frank, K B; Cheng, Y C

1986-02-05

Purine ribonucleoside monophosphates were found to inhibit chain elongation catalyzed by herpes simplex virus (HSV) DNA polymerase when DNA template-primer concentrations were rate-limiting. Inhibition was fully competitive with DNA template-primer during chain elongation; however, DNA polymerase-associated exonuclease activity was inhibited noncompetitively with respect to DNA. Combinations of 5'-GMP and phosphonoformate were kinetically mutually exclusive in dual inhibitor studies. Pyrimidine nucleoside monophosphates and deoxynucleoside monophosphates were less inhibitory than purine riboside monophosphates. The monophosphates of 9-beta-D-arabinofuranosyladenine, Virazole (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide), 9-(2-hydroxyethoxymethyl)guanine, and 9-(1,3-dihydroxy-2-propoxymethyl)guanine exerted little or no inhibition. In contrast to HSV DNA polymerase, human DNA polymerase alpha was not inhibited by purine ribonucleoside monophosphates. These studies suggest the possibility of a physiological role of purine ribonucleoside monophosphates as regulators of herpesvirus DNA synthesis and a new approach to developing selective anti-herpesvirus compounds.

Axolotl hemoglobin: cDNA-derived amino acid sequences of two alpha globins and a beta globin from an adult Ambystoma mexicanum.

PubMed

Shishikura, Fumio; Takeuchi, Hiro-aki; Nagai, Takatoshi

2005-11-01

Erythrocytes of the adult axolotl, Ambystoma mexicanum, have multiple hemoglobins. We separated and purified two kinds of hemoglobin, termed major hemoglobin (Hb M) and minor hemoglobin (Hb m), from a five-year-old male by hydrophobic interaction column chromatography on Alkyl Superose. The hemoglobins have two distinct alpha type globin polypeptides (alphaM and alpham) and a common beta globin polypeptide, all of which were purified in FPLC on a reversed-phase column after S-pyridylethylation. The complete amino acid sequences of the three globin chains were determined separately using nucleotide sequencing with the assistance of protein sequencing. The mature globin molecules were composed of 141 amino acid residues for alphaM globin, 143 for alpham globin and 146 for beta globin. Comparing primary structures of the five kinds of axolotl globins, including two previously established alpha type globins from the same species, with other known globins of amphibians and representatives of other vertebrates, we constructed phylogenetic trees for amphibian hemoglobins and tetrapod hemoglobins. The molecular trees indicated that alphaM, alpham, beta and the previously known alpha major globin were adult types of globins and the other known alpha globin was a larval type. The existence of two to four more globins in the axolotl erythrocyte is predicted.

Cloning and expression of a novel UDP-GlcNAc:alpha-D-mannoside beta1,2-N-acetylglucosaminyltransferase homologous to UDP-GlcNAc:alpha-3-D-mannoside beta1,2-N-acetylglucosaminyltransferase I.

PubMed Central

Zhang, Wenli; Betel, Doron; Schachter, Harry

2002-01-01

A TBLASTN search with human UDP-GlcNAc:alpha-3-d-mannoside beta-1,2-N-acetylglucosaminyltransferase I (GnT I; EC 2.4.1.101) as a probe identified human and mouse Unigenes encoding a protein similar to human GnT I (34% identity over 340 amino acids). The recombinant protein converted Man(alpha1-6)[Man(alpha1-3)]Man(beta1-)O-octyl to Man(alpha1-6)[GlcNAc(beta1-2)Man(alpha1-3)]Man(beta1-)O-octyl, the reaction catalysed by GnT I. The enzyme also added GlcNAc to Man(alpha1-6)[GlcNAc(beta1-2)Man(alpha1-3)]Man(beta1-)O-octyl (the substrate for beta-1,2-N-acetylglucosaminyltransferase II), Man(alpha1-)O-benzyl [with K(m) values of approximately 0.3 and >30 mM for UDP-GlcNAc and Man(alpha1-)O-benzyl respectively] and the glycopeptide CYA[Man(alpha1-)O-T]AV (K(m) approximately 12 mM). The product formed with Man(alpha1-)O-benzyl was identified as GlcNAc(beta1-2)Man(alpha1-)O-benzyl by proton NMR spectroscopy. The enzyme was named UDP-GlcNAc:alpha-d-mannoside beta-1,2-N-acetylglucosaminyltransferase I.2 (GnT I.2). The human gene mapped to chromosome 1. Northern-blot analysis showed a 3.3 kb message with a wide tissue distribution. The cDNA has a 1980 bp open reading frame encoding a 660 amino acid protein with a type-2 domain structure typical of glycosyltransferases. Man(beta1-)O-octyl, Man(beta1-)O-p-nitrophenyl and GlcNAc(beta1-2)Man(alpha1-6)[GlcNAc(beta1-2)Man(alpha1-3)]Man(beta1-4)GlcNAc(beta1-4)GlcNAc(beta1-)O-Asn were not acceptors, indicating that GnT I.2 is specific for alpha-linked terminal Man and does not have N-acetylglucosaminyltransferase III, IV, V, VII or VIII activities. CYA[Man(alpha1-)O-T]AV was between three and seven times more effective as an acceptor than the other substrates, suggesting that GnT I.2 may be responsible for the synthesis of the GlcNAc(beta1-2)Man(alpha1-)O-Ser/Thr moiety on alpha-dystroglycan and other O-mannosylated proteins. PMID:11742540

Cancer-linked satellite 2 DNA hypomethylation does not regulate Sat2 non-coding RNA expression and is initiated by heat shock pathway activation.

PubMed

Tilman, Gaëlle; Arnoult, Nausica; Lenglez, Sandrine; Van Beneden, Amandine; Loriot, Axelle; De Smet, Charles; Decottignies, Anabelle

2012-08-01

Epigenetic dysfunctions, including DNA methylation alterations, play major roles in cancer initiation and progression. Although it is well established that gene promoter demethylation activates transcription, it remains unclear whether hypomethylation of repetitive heterochromatin similarly affects expression of non-coding RNA from these loci. Understanding how repetitive non-coding RNAs are transcriptionally regulated is important given that their established upregulation by the heat shock (HS) pathway suggests important functions in cellular response to stress, possibly by promoting heterochromatin reconstruction. We found that, although pericentromeric satellite 2 (Sat2) DNA hypomethylation is detected in a majority of cancer cell lines of various origins, DNA methylation loss does not constitutively hyperactivate Sat2 expression, and also does not facilitate Sat2 transcriptional induction upon heat shock. In melanoma tumor samples, our analysis revealed that the HS response, frequently upregulated in tumors, is probably the main determinant of Sat2 RNA expression in vivo. Next, we tested whether HS pathway hyperactivation may drive Sat2 demethylation. Strikingly, we found that both hyperthermia and hyperactivated RasV12 oncogene, another potent inducer of the HS pathway, reduced Sat2 methylation levels by up to 27% in human fibroblasts recovering from stress. Demethylation occurred locally on Sat2 repeats, resulting in a demethylation signature that was also detected in cancer cell lines with moderate genome-wide hypomethylation. We therefore propose that upregulation of Sat2 transcription in response to HS pathway hyperactivation during tumorigenesis may promote localized demethylation of the locus. This, in turn, may contribute to tumorigenesis, as demethylation of Sat2 was previously reported to favor chromosomal rearrangements.

DNA synthesis arrest sites at the right terminus of rat long interspersed repeated (LINE or L1Rn) DNA family members.

PubMed Central

d'Ambrosio, E; Furano, A V

1987-01-01

An approximately equal to 150-bp GC-rich (approximately equal to 60%) region is at the right end of rat long interspersed repeated DNA (LINE or L1Rn) family members. We report here that one of the DNA strands from this region contains several non-palindromic sites that strongly arrest DNA synthesis in vitro by the prokaryotic Klenow and T4 DNA polymerases, the eukaryotic alpha polymerase, and AMV reverse transcriptase. The strongest arrest sites are G-rich (approximately equal to 70%) homopurine stretches of 18 or more residues. Shorter homopurine stretches (12 residues or fewer) did not arrest DNA synthesis even if the stretch contains 11/12 G residues. Arrest of the prokaryotic polymerases was not affected by their respective single strand binding proteins or polymerase accessory proteins. The region of duplex DNA which contains DNA synthesis arrest sites reacts with bromoacetaldehyde when present in negatively supercoiled molecules. By contrast, homopurine stretches that do not arrest DNA synthesis do not react with bromoacetaldehyde. The presence of bromoacetaldehyde-reactive bases in a G-rich homopurine-containing duplex under torsional stress is thought to be caused by base stacking in the homopurine strand. Therefore, we suggest that base-stacked regions of the template arrest DNA synthesis. Images PMID:2436148

Effects of alpha/beta-androstenediol immune regulating hormones on bone remodeling and apoptosis in osteoblasts.

PubMed

Urban, Nicole H; Chamberlin, Brett; Ramage, Samuel; Roberts, Zachary; Loria, Roger M; Beckman, Matthew J

2008-06-01

A large body of evidence suggests that the immune system directly impacts bone physiology. We tested whether immune regulating hormones (IRH), 17beta-androstenediol (beta-AED), 7beta,17beta-androstenetriol (beta-AET) or the 17alpha-androstenediol (alpha-AED), and 7alpha,17beta-androstenetriol (alpha-AET) metabolites could directly influence bone remodeling in vitro using human fetal osteoblasts (FOB-9). The impact on bone remodeling was examined by comparing the ratio of RANKL/OPG gene expression in response to AED and AET compounds. The alpha-AED was found to significantly increase in the ratio of RANKL/OPG gene expression and altering the morphology of RANKL stained FOB-9 cells. Cell viability was assessed using a Live/Dead assay. Again alpha-AED was unique in its ability to reduce the proportion of viable cells, and to induce mild apoptosis of FOB-9 cells. Treatment of FOB-9 cells with WY14643, an activator of PPAR-alpha and -gamma, also significantly elevated the percentage of dead cells. This increase was abolished by co-treatment with GW9962, a specific inhibitor of PPAR-gamma. Analysis of PPAR-gamma mRNA by Quantitative RT-PCR and its activation by DNA binding demonstrated that alpha-AED increased PPAR-gamma activation by 19%, while beta-AED conferred a 37% decrease in PPAR-gamma activation. In conclusion, alpha-AED opposed beta-AED by elevating a bone resorption scenario in osteoblast cells. The increase in RANKL/OPG is modulated by an activation of PPAR-gamma that in turn caused mild apoptosis of FOB-9 cells.

Mutations in the PCCA gene encoding the {alpha} subunit of propionyl-CoA carboxylase in patients with propionic acidemia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Campeau, E.; Leon-Del-Rio, A.; Gravel, R.A.

Propionic acidemia is a rare autosomal recessive disorder characterized by a deficiency of the mitochondrial biotin-dependent enzyme, propionyl-CoA carboxylase (PCC). PCC has the structure {alpha}{sub 4}{beta}{sub 4}, with the {alpha} subunit containing the biotin prosthetic group. This study is concerned with defining the spectrum of mutations occurring in the PCCA gene encoding the {alpha} subunit. Mutations were initially assigned to this gene through complementation experiments done after somatic fusion of patient fibroblasts. The analyses were performed on PCR-amplified reverse transcripts of fibroblast RNA. The mutations were identified by single strand conformational polymorphism analysis and direct sequencing of PCR products. Threemore » candidate disease-causing mutations and one DNA polymorphism were identified in the {alpha} subunit sequence in different patients: (1) a 3 bp deletion {triangle}CTG{sub 2058-2060}, which eliminates Cys687 near the biotin binding site (Lys669); (2) T{sub 611}{r_arrow}A which converts Met204 to Lys in a highly conserved region matching that of an ATP binding site; (3) An {approximately}50 bp deletion near the 3{prime} end of the cDNA which likely corresponds to the loss of an exon due to a splicing defect; and (4) a 3 bp insertion, +CAG{sub 2203}, located downstream of the stop codon, which is likely a DNA polymorphism. In order to determine the effect of the Cys687 deletion on the biotinylation of PCC, we expressed the mutation in a 67 amino acid C-terminal fragment of the PCC {alpha} subunit in E. coli in which biotinylation is directed by the bacterial biotin ligase. While the mutant peptide was expressed at about half-normal levels, the biotinylation of the peptide that was present was reduced to only {approximately}20% normal. We suggest, therefore, that the absence of PCC activity due to {triangle}Cys687 results at least in part from defective biotinylation of an unstable protein.« less

Mesospheric Simulations with the NOGAPS-ALPHA model: Applications to the Summer Polar Mesosphere and AIM data

NASA Astrophysics Data System (ADS)

Siskind, D. E.; Eckermann, S. D.; McCormack, J. P.; Hoppel, K. W.; Russell, J. M.; Bailey, S.; Hervig, M.; Rusch, D.

2007-12-01

The Navy Operational Global Atmospheric Prediction System (NOGAPS), the Department of Defense's global numerical weather prediction (NWP) system, consists of two main components: the Naval Research Laboratory (NRL) Atmospheric Variational Data Assimilation System (NAVDAS) and a global spectral general circulation model (GCM) for forecasting. NRL researchers are currently developing an Advanced-Level Physics High-Altitude (ALPHA) NOGAPS prototype that extends the currently operational 1 hPa upper boundary of NOGAPS through the mesosphere and lower thermosphere (MLT) to ~110 km. We report results of preliminary experiments with this NOGAPS-ALPHA system during May-June 2007, focused on the northern hemisphere (NH) summer mesosphere observed from the Aeronomy of Ice in the Mesosphere (AIM) satellite. These AIM-period NOGAPS-ALPHA experiments have two main goals: to provide global modeling support for AIM science and to allow objective validation of these new NOGAPS-ALPHA MLT fields using independent observations from AIM. We report results of runs which assimilate temperature and water vapor data from the SABER and MLS instruments up to ~0.01 hPa. We investigate the development of the cold NH summer mesopause in NOGAPS-ALPHA and its sensitivity to parameterized nonorographic gravity wave drag (GWD) and radiative heating/cooling by comparing with temperatures and water vapor measured by AIM's SOFIE instrument. We can also compare the variability in the NOGAPS-ALPHA temperature and water vapor fields with mesospheric cloud occurrence statistics measured by CIPS on AIM.

Construction and properties of a cell line constitutively expressing the herpes simplex virus glycoprotein B dependent on functional alpha 4 protein synthesis.

PubMed Central

Arsenakis, M; Hubenthal-Voss, J; Campadelli-Fiume, G; Pereira, L; Roizman, B

1986-01-01

We report the construction of a cell line constitutively expressing the glycoprotein B (gB) of herpes simplex virus (HSV) 1. The cell line was constructed in two steps. In the first, a baby hamster kidney cell line was transfected with the DNA of a plasmid containing the neomycin phosphotransferase gene that confers resistance to the antibiotic G418 and the gene specifying a temperature-sensitive (ts-) alpha 4 protein of HSV-1, the major viral regulatory protein. A clonal cell line, alpha 4/c113, selected for resistance to the antibiotic G418, expressed high levels of alpha 4 protein constitutively. Superinfection of these cells with HSV-2 resulted in twofold induction of the resident HSV-1 alpha 4 gene. In the second step, alpha 4/c113 cells were transfected with the DNA of a plasmid carrying the gB gene and the mouse methotrexate resistance dihydrofolate reductase gene. A clonal cell line, alpha 4/c113/gB, selected for methotrexate resistance expressed gB constitutively. Expression of both gB and alpha 4 continued unabated for at least 32 serial passages. Cells passaged serially in medium containing both methotrexate and G418 after passage 10 contained a higher copy number of the alpha 4 gene and produced larger amounts of both gB and alpha 4 proteins than did cells maintained in medium containing methotrexate alone. Expression of gB was dependent on the presence of functional alpha 4 protein inasmuch as expression of gB ceased on shift up to nonpermissive temperatures, when shifted to permissive temperatures, the cell line reinitiated expression of gB after a delay commensurate with the length of incubation at the nonpermissive temperature, and the cell-resident HSV-1 gB gene was expressed at the nonpermissive temperature in cells infected with a recombinant expressing a ts+ alpha 4 protein and an HSV-2 gB. The properties of the alpha 4/c113 cell line suggest that it may express other viral genes induced by alpha 4 protein constitutively, provided that the

THE LYMAN ALPHA REFERENCE SAMPLE: EXTENDED LYMAN ALPHA HALOS PRODUCED AT LOW DUST CONTENT

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hayes, Matthew; Oestlin, Goeran; Duval, Florent

2013-03-10

We report on new imaging observations of the Lyman alpha emission line (Ly{alpha}), performed with the Hubble Space Telescope, that comprise the backbone of the Lyman alpha Reference Sample. We present images of 14 starburst galaxies at redshifts 0.028 < z < 0.18 in continuum-subtracted Ly{alpha}, H{alpha}, and the far ultraviolet continuum. We show that Ly{alpha} is emitted on scales that systematically exceed those of the massive stellar population and recombination nebulae: as measured by the Petrosian 20% radius, R{sub P20}, Ly{alpha} radii are larger than those of H{alpha} by factors ranging from 1 to 3.6, with an average ofmore » 2.4. The average ratio of Ly{alpha}-to-FUV radii is 2.9. This suggests that much of the Ly{alpha} light is pushed to large radii by resonance scattering. Defining the Relative Petrosian Extension of Ly{alpha} compared to H{alpha}, {xi}{sub Ly{alpha}} = R {sup Ly{alpha}}{sub P20}/R {sup H{alpha}}{sub P20}, we find {xi}{sub Ly{alpha}} to be uncorrelated with total Ly{alpha} luminosity. However, {xi}{sub Ly{alpha}} is strongly correlated with quantities that scale with dust content, in the sense that a low dust abundance is a necessary requirement (although not the only one) in order to spread Ly{alpha} photons throughout the interstellar medium and drive a large extended Ly{alpha} halo.« less

Beams of protons and alpha particles greater than approximately 30 keV/charge from the earth's bow shock

NASA Technical Reports Server (NTRS)

Scholer, M.; Ipavich, F. M.; Gloeckler, G.

1981-01-01

Two beamlike particle events (30 keV/charge to 160 keV/charge) upstream of the earth's bow shock have been investigated with the Max-Planck-Institut/University of Maryland ultralow energy and charge analyzer on ISEE 1. These beams consist of protons as well as of alpha particles, and the spectra are generally steep and are decreasing with increasing energy. During one event the spectra of both protons and alpha particles have a maximum at approximately 65 keV/charge. During these events, the interplanetary magnetic field through the satellite position was almost tangent to the bow shock, and application of the theory of acceleration predicts acceleration of a solar wind particle up to 60 keV/nucleon in a single reflection. The observation of reflected protons as well as alpha particles has implications for the physical reflection process usually not discussed in acceleration theories.

Functional interaction of the DNA-binding transcription factor Sp1 through its DNA-binding domain with the histone chaperone TAF-I.

PubMed

Suzuki, Toru; Muto, Shinsuke; Miyamoto, Saku; Aizawa, Kenichi; Horikoshi, Masami; Nagai, Ryozo

2003-08-01

Transcription involves molecular interactions between general and regulatory transcription factors with further regulation by protein-protein interactions (e.g. transcriptional cofactors). Here we describe functional interaction between DNA-binding transcription factor and histone chaperone. Affinity purification of factors interacting with the DNA-binding domain of the transcription factor Sp1 showed Sp1 to interact with the histone chaperone TAF-I, both alpha and beta isoforms. This interaction was specific as Sp1 did not interact with another histone chaperone CIA nor did other tested DNA-binding regulatory factors (MyoD, NFkappaB, p53) interact with TAF-I. Interaction of Sp1 and TAF-I occurs both in vitro and in vivo. Interaction with TAF-I results in inhibition of DNA-binding, and also likely as a result of such, inhibition of promoter activation by Sp1. Collectively, we describe interaction between DNA-binding transcription factor and histone chaperone which results in negative regulation of the former. This novel regulatory interaction advances our understanding of the mechanisms of eukaryotic transcription through DNA-binding regulatory transcription factors by protein-protein interactions, and also shows the DNA-binding domain to mediate important regulatory interactions.

Hb Adana (HBA2 or HBA1: c.179G > A) and alpha thalassemia: Genotype-phenotype correlation.

PubMed

Singh, Sharon A; Sarangi, Susmita; Appiah-Kubi, Abena; Hsu, Peihong; Smith, W Byron; Gallagher, Patrick G; Glader, Bertil; Chui, David H K

2018-05-11

Alpha thalassemia due to nondeletional mutations usually leads to more severe disease than that caused by deletional mutations. Devastating outcomes such as hydrops fetalis can occur with two nondeletional mutations, therefore warranting DNA-based workup for suspected carriers with subtle hematological abnormalities for family counseling purposes. We describe three cases with hemoglobin (Hb) Adana, a nondeletional alpha chain mutation, compounded with an alpha globin gene deletion resulting in thalassemia intermedia. We review the literature, draw genotype-phenotype correlations from published cases of Hb Adana, and propose that this correlation can be used by clinicians to help direct diagnostic studies and urge hematologists to thoroughly workup high-risk patients. © 2018 Wiley Periodicals, Inc.

DNA hypomethylation of individual sequences in aborted cloned bovine fetuses.

PubMed

Chen, Tao; Jiang, Yan; Zhang, Yan-Ling; Liu, Jing-He; Hou, Yi; Schatten, Heide; Chen, Da-Yuan; Sun, Qing-Yuan

2005-09-01

Cloned bovines have a much higher abortion rate than those derived in vivo. Available evidence indicates that inappropriate epigenetic reprogramming of donor nuclei is the primary cause of cloning failure. To gain a better understanding of the DNA methylation changes associated with the high abortion rate of cloned bovines, we examined the DNA methylation status of a repeated sequence (satellite I) and the promoter regions of two single-copy genes (interleukin 3/cytokeratin) in aborted cloned fetuses, aborted fetuses derived from artificial insemination (AI), cloned adults and AI adults by bisulfite sequencing and restriction enzyme analysis. Two of four aborted cloned fetuses show very low methylation levels in the two single-copy gene promoter regions. One of the two fetuses also showed undermethylated status in the satellite I sequence. The other two aborted cloned fetuses have similar methylation levels to those of aborted AI fetuses. However, no difference in methylation was observed between cloned adults and AI adults. Our results demonstrate for the first time the undermethylated status of individual sequences in aborted cloned fetuses. These findings suggest that aberrant DNA methylation may contribute to the developmental failure of cloned bovine fetuses.

Integrated Satellite Control in REIMEI (INDEX) Satellite

NASA Astrophysics Data System (ADS)

Fukuda, Seisuke; Mizuno, Takahide; Sakai, Shin-Ichiro; Fukushima, Yousuke; Saito, Hirobumi

REIMEI/INDEX (INnovative-technology Demonstration EXperiment) is a 70kg class small satellite which the Institute of Space and Astronautical Science, Japan Exploration Agency, ISAS/JAXA, has developed for observation of auroral small-scale dynamics as well as demonstration of advanced satellite technologies. An important engineering mission of REIMEI is integrated satellite control using commercial RISC CPUs with a triple voting system in order to ensure fault-tolerance against radiation hazards. Software modules concerning every satellite function, such as attitude control, data handling, and mission applications, work cooperatively so that highly sophisticated satellite control can be performed. In this paper, after a concept of the integrated satellite control is introduced, the Integrated Controller Unit (ICU) is described in detail. Also unique topics in developing the integrated control system are shown.

W-enriched satellite sequence in the Indian meal moth, Plodia interpunctella (Lepidoptera, Pyralidae).

PubMed

Dalíková, Martina; Zrzavá, Magda; Kubíčková, Svatava; Marec, František

2017-10-01

The W chromosome of most lepidopteran species represents the largest heterochromatin entity in the female genome. Although satellite DNA is a typical component of constitutive heterochromatin, there are only a few known satellite DNAs (satDNAs) located on the W chromosome in moths and butterflies. In this study, we isolated and characterized new satDNA (PiSAT1) from microdissected W chromosomes of the Indian meal moth, Plodia interpunctella. Even though the PiSAT1 is mainly localized near the female-specific segment of the W chromosome, short arrays of this satDNA also occur on autosomes and/or the Z chromosome. Probably due to the predominant location in the non-recombining part of the genome, PiSAT1 exhibits a relatively large nucleotide variability in its monomers. However, at least a part of all predicted functional motifs is located in conserved regions. Moreover, we detected polyadenylated transcripts of PiSAT1 in all developmental stages and in both sexes (female and male larvae, pupae and adults). Our results suggest a potential structural and functional role of PiSAT1 in the P. interpunctella genome, which is consistent with accumulating evidence for the important role of satDNAs in eukaryotic genomes.

Schedule-dependent inhibition of hypoxia-inducible factor-1alpha protein accumulation, angiogenesis, and tumor growth by topotecan in U251-HRE glioblastoma xenografts.

PubMed

Rapisarda, Annamaria; Zalek, Jessica; Hollingshead, Melinda; Braunschweig, Till; Uranchimeg, Badarch; Bonomi, Carrie A; Borgel, Suzanne D; Carter, John P; Hewitt, Stephen M; Shoemaker, Robert H; Melillo, Giovanni

2004-10-01

We have previously shown that topotecan, a topoisomerase I poison, inhibits hypoxia-inducible factor (HIF)-1alpha protein accumulation by a DNA damage-independent mechanism. Here, we report that daily administration of topotecan inhibits HIF-1alpha protein expression in U251-HRE glioblastoma xenografts. Concomitant with HIF-1alpha inhibition, topotecan caused a significant tumor growth inhibition associated with a marked decrease of angiogenesis and expression of HIF-1 target genes in tumor tissue. These results provide a compelling rationale for testing topotecan in clinical trials to target HIF-1 in cancer patients.

The mongoose acetylcholine receptor alpha-subunit: analysis of glycosylation and alpha-bungarotoxin binding.

PubMed

Asher, O; Jensen, B S; Lupu-Meiri, M; Oron, Y; Fuchs, S

1998-04-17

The mongoose AChR alpha-subunit has been cloned and shown to be highly homologous to other AChR alpha-subunits, with only six differences in amino acid residues at positions that are conserved in animal species that bind alpha-bungarotoxin (alpha-BTX). Four of these six substitutions cluster in the ligand binding site, and one of them, Asn-187, forms a consensus N-glycosylation site. The mongoose glycosylated alpha-subunit has a higher apparent molecular mass than that of the rat glycosylated alpha-subunit, probably resulting from the additional glycosylation at Asn-187 of the mongoose subunit. The in vitro translated mongoose alpha-subunit, in a glycosylated or non-glycosylated form, does not bind alpha-BTX, indicating that lack of alpha-BTX binding can be achieved also in the absence of glycosylation.

Parallel nucleic acid recognition by the LNA (locked nucleic acid) stereoisomers beta-L-LNA and alpha-D-LNA; studies in the mirror image world.

PubMed

Christensen, Nanna K; Bryld, Torsten; Sørensen, Mads D; Arar, Khalil; Wengel, Jesper; Nielsen, Poul

2004-02-07

Two LNA (locked nucleic acid) stereoisomers (beta-L-LNA and alpha-D-LNA) are evaluated in the mirror-image world, that is by the study of two mixed sequences of LNA and alpha-L-LNA and their L-DNA and L-RNA complements. Both are found to display high-affinity RNA-recognition by the formation of duplexes with parallel strand orientation.

Anomalous Temporal Behaviour of Broadband Ly Alpha Observations During Solar Flares from SDO/EVE

NASA Technical Reports Server (NTRS)

Milligan, Ryan O.; Chamberlin, Phillip C.

2016-01-01

broadband Ly-alpha observations of solar flares. Comparisons have also been made with other broadband Ly-alpha photometers such as PROBA2 (Project for On-Board Autonomy-2) / LYRA (Lyman Alpha Radiometer) and GOES (Geostationary Operational Environmental Satellite) / EUVE (Extreme Ultraviolet Explorer).

Cytotoxicity and apoptotic activities of alpha-, gamma- and delta-tocotrienol isomers on human cancer cells.

PubMed

Lim, Su-Wen; Loh, Hwei-San; Ting, Kang-Nee; Bradshaw, Tracey D; Zeenathul, Nazariah A

2014-12-06

Tocotrienols, especially the gamma isomer was discovered to possess cytotoxic effects associated with the induction of apoptosis in numerous cancers. Individual tocotrienol isomers are believed to induce dissimilar apoptotic mechanisms in different cancer types. This study was aimed to compare the cytotoxic potency of alpha-, gamma- and delta-tocotrienols, and to explore their resultant apoptotic mechanisms in human lung adenocarcinoma A549 and glioblastoma U87MG cells which are scarcely researched. The cytotoxic effects of alpha-, gamma- and delta-tocotrienols in both A549 and U87MG cancer cells were first determined at the cell viability and morphological aspects. DNA damage types were then identified by comet assay and flow cytometric study was carried out to support the incidence of apoptosis. The involvements of caspase-8, Bid, Bax and mitochondrial membrane permeability (MMP) in the execution of apoptosis were further expounded. All tocotrienols inhibited the growth of A549 and U87MG cancer cells in a concentration- and time-dependent manner. These treated cancer cells demonstrated some hallmarks of apoptotic morphologies, apoptosis was further confirmed by cell accumulation at the pre-G1 stage. All tocotrienols induced only double strand DNA breaks (DSBs) and no single strand DNA breaks (SSBs) in both treated cancer cells. Activation of caspase-8 leading to increased levels of Bid and Bax as well as cytochrome c release attributed by the disruption of mitochondrial membrane permeability in both A549 and U87MG cells were evident. This study has shown that delta-tocotrienol, in all experimental approaches, possessed a higher efficacy (shorter induction period) and effectiveness (higher induction rate) in the execution of apoptosis in both A549 and U87MG cancer cells as compared to alpha- and gamma-tocotrienols. Tocotrienols in particular the delta isomer can be an alternative chemotherapeutic agent for treating lung and brain cancers.

Comparative molecular cytogenetic analyses of a major tandemly repeated DNA family and retrotransposon sequences in cultivated jute Corchorus species (Malvaceae).

PubMed

Begum, Rabeya; Zakrzewski, Falk; Menzel, Gerhard; Weber, Beatrice; Alam, Sheikh Shamimul; Schmidt, Thomas

2013-07-01

The cultivated jute species Corchorus olitorius and Corchorus capsularis are important fibre crops. The analysis of repetitive DNA sequences, comprising a major part of plant genomes, has not been carried out in jute but is useful to investigate the long-range organization of chromosomes. The aim of this study was the identification of repetitive DNA sequences to facilitate comparative molecular and cytogenetic studies of two jute cultivars and to develop a fluorescent in situ hybridization (FISH) karyotype for chromosome identification. A plasmid library was generated from C. olitorius and C. capsularis with genomic restriction fragments of 100-500 bp, which was complemented by targeted cloning of satellite DNA by PCR. The diversity of the repetitive DNA families was analysed comparatively. The genomic abundance and chromosomal localization of different repeat classes were investigated by Southern analysis and FISH, respectively. The cytosine methylation of satellite arrays was studied by immunolabelling. Major satellite repeats and retrotransposons have been identified from C. olitorius and C. capsularis. The satellite family CoSat I forms two undermethylated species-specific subfamilies, while the long terminal repeat (LTR) retrotransposons CoRetro I and CoRetro II show similarity to the Metaviridea of plant retroelements. FISH karyotypes were developed by multicolour FISH using these repetitive DNA sequences in combination with 5S and 18S-5·8S-25S rRNA genes which enable the unequivocal chromosome discrimination in both jute species. The analysis of the structure and diversity of the repeated DNA is crucial for genome sequence annotation. The reference karyotypes will be useful for breeding of jute and provide the basis for karyotyping homeologous chromosomes of wild jute species to reveal the genetic and evolutionary relationship between cultivated and wild Corchorus species.

Comparative molecular cytogenetic analyses of a major tandemly repeated DNA family and retrotransposon sequences in cultivated jute Corchorus species (Malvaceae)

PubMed Central

Begum, Rabeya; Zakrzewski, Falk; Menzel, Gerhard; Weber, Beatrice; Alam, Sheikh Shamimul; Schmidt, Thomas

2013-01-01

Background and Aims The cultivated jute species Corchorus olitorius and Corchorus capsularis are important fibre crops. The analysis of repetitive DNA sequences, comprising a major part of plant genomes, has not been carried out in jute but is useful to investigate the long-range organization of chromosomes. The aim of this study was the identification of repetitive DNA sequences to facilitate comparative molecular and cytogenetic studies of two jute cultivars and to develop a fluorescent in situ hybridization (FISH) karyotype for chromosome identification. Methods A plasmid library was generated from C. olitorius and C. capsularis with genomic restriction fragments of 100–500 bp, which was complemented by targeted cloning of satellite DNA by PCR. The diversity of the repetitive DNA families was analysed comparatively. The genomic abundance and chromosomal localization of different repeat classes were investigated by Southern analysis and FISH, respectively. The cytosine methylation of satellite arrays was studied by immunolabelling. Key Results Major satellite repeats and retrotransposons have been identified from C. olitorius and C. capsularis. The satellite family CoSat I forms two undermethylated species-specific subfamilies, while the long terminal repeat (LTR) retrotransposons CoRetro I and CoRetro II show similarity to the Metaviridea of plant retroelements. FISH karyotypes were developed by multicolour FISH using these repetitive DNA sequences in combination with 5S and 18S–5·8S–25S rRNA genes which enable the unequivocal chromosome discrimination in both jute species. Conclusions The analysis of the structure and diversity of the repeated DNA is crucial for genome sequence annotation. The reference karyotypes will be useful for breeding of jute and provide the basis for karyotyping homeologous chromosomes of wild jute species to reveal the genetic and evolutionary relationship between cultivated and wild Corchorus species. PMID:23666888

Regulation of gene expression by dietary Ca2+ in kidneys of 25-hydroxyvitamin D3-1 alpha-hydroxylase knockout mice.

PubMed

Hoenderop, Joost G J; Chon, Helena; Gkika, Dimitra; Bluyssen, Hans A R; Holstege, Frank C P; St-Arnaud, Rene; Braam, Branko; Bindels, Rene J M

2004-02-01

Pseudovitamin D deficiency rickets (PDDR) is an autosomal disease, characterized by undetectable levels of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), rickets and secondary hyperparathyroidism. Mice in which the 25-hydroxyvitamin D3-1 alpha-hydroxylase (1 alpha-OHase) gene was inactivated, presented the same clinical phenotype as patients with PDDR. cDNA Microarray technology was used on kidneys of 1 alpha-OHase knockout mice to study the expression profile of renal genes in this Ca2+-related disorder. Genome wide molecular events that occur during the rescue of these mice by high dietary Ca2+ intake were studied by the use of 15K cDNA microarray chips. 1 alpha-OHase knockout mice fed a normal Ca2+ diet developed severe hypocalcemia, rickets and died with an average life span of 12 +/- 2 weeks. Intriguingly, 1 alpha-OHase-/- mice supplemented with an enriched Ca2+ diet were normocalcemic and not significantly different from wild-type mice. Inactivation of the 1 alpha-OHase gene resulted in a significant regulation of +/- 1000 genes, whereas dietary Ca2+ supplementation of the 1 alpha-OHase-/- mice revealed +/- 2000 controlled genes. Interestingly, 557 transcripts were regulated in both situations implicating the involvement in the dietary Ca2+-mediated rescue mechanism of the 1 alpha-OHase-/- mice. Conspicuous regulated genes encoded for signaling molecules like the PDZ-domain containing protein channel interacting protein, FK binding protein type 4, kinases, and importantly Ca2+ transporting proteins including the Na+-Ca2+ exchanger, calbindin-D28K and the Ca2+ sensor calmodulin. Dietary Ca2+ intake normalized disturbances in the Ca2+ homeostasis due to vitamin D deficiency that were accompanied by the regulation of a subset of renal genes, including well-known renal Ca2+ transport protein genes, but also genes not previously identified as playing a role in renal Ca2+ handling.

40 CFR 721.10300 - Benzeneacetic acid, .alpha.-chloro-.alpha.-phenyl-, ethyl ester.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Benzeneacetic acid, .alpha.-chloro-.alpha.-phenyl-, ethyl ester. 721.10300 Section 721.10300 Protection of Environment ENVIRONMENTAL... Significant New Uses for Specific Chemical Substances § 721.10300 Benzeneacetic acid, .alpha.-chloro-.alpha...

40 CFR 721.10300 - Benzeneacetic acid, .alpha.-chloro-.alpha.-phenyl-, ethyl ester.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Benzeneacetic acid, .alpha.-chloro-.alpha.-phenyl-, ethyl ester. 721.10300 Section 721.10300 Protection of Environment ENVIRONMENTAL... Significant New Uses for Specific Chemical Substances § 721.10300 Benzeneacetic acid, .alpha.-chloro-.alpha...

40 CFR 721.10300 - Benzeneacetic acid, .alpha.-chloro-.alpha.-phenyl-, ethyl ester.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Benzeneacetic acid, .alpha.-chloro-.alpha.-phenyl-, ethyl ester. 721.10300 Section 721.10300 Protection of Environment ENVIRONMENTAL... Significant New Uses for Specific Chemical Substances § 721.10300 Benzeneacetic acid, .alpha.-chloro-.alpha...

Omentin inhibits TNF-{alpha}-induced expression of adhesion molecules in endothelial cells via ERK/NF-{kappa}B pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhong, Xia, E-mail: zhongxia1977@126.com; Li, Xiaonan; Liu, Fuli

2012-08-24

Highlights: Black-Right-Pointing-Pointer Omentin inhibited TNF-{alpha}-induced adhesion of THP-1 cells to HUVECs. Black-Right-Pointing-Pointer Omentin reduces expression of ICAM-1 and VCAM-1 induced by TNF-{alpha} in HUVECs. Black-Right-Pointing-Pointer Omentin inhibits TNF-{alpha}-induced ERK and NF-{kappa}B activation in HUVECs. Black-Right-Pointing-Pointer Omentin supreeses TNF-{alpha}-induced expression of ICAM-1 and VCAM-1 via ERK/NF-{kappa}B pathway. -- Abstract: In the present study, we investigated whether omentin affected the expression of intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in tumor necrosis factor-{alpha} (TNF-{alpha}) induced human umbilical vein endothelial cells (HUVECs). Our data showed that omentin decreased TNF-{alpha}-induced expression of ICAM-1 and VCAM-1 in HUVECs. In addition, omentin inhibitedmore » TNF-{alpha}-induced adhesion of THP-1 cells to HUVECs. Further, we found that omentin inhibited TNF-{alpha}-activated signal pathway of nuclear factor-{kappa}B (NF-{kappa}B) by preventing NF-{kappa}B inhibitory protein (I{kappa}B{alpha}) degradation and NF-{kappa}B/DNA binding activity. Omentin pretreatment significantly inhibited TNF-{alpha}-induced ERK activity and ERK phosphorylation in HUVECs. Pretreatment with PD98059 suppressed TNF-{alpha}-induced NF-{kappa}B activity. Omentin, NF-kB inhibitor (BAY11-7082) and ERK inhibitor (PD98059) reduced the up-regulation of ICAM-1 and VCAM-1 induced by TNF-{alpha}. These results suggest that omentin may inhibit TNF-{alpha}-induced expression of adhesion molecules in endothelial cells via blocking ERK/NF-{kappa}B pathway.« less

The Val192Leu mutation in the alpha-subunit of beta-hexosaminidase A is not associated with the B1-variant form of Tay-Sachs disease.

PubMed

Hou, Y; Vavougios, G; Hinek, A; Wu, K K; Hechtman, P; Kaplan, F; Mahuran, D J

1996-07-01

Substitution mutations adversely affecting the alpha-subunit of beta-hexosaminidase A (alphabeta) (EC 3.2.1.52) result in Tay-Sachs disease. The majority affect the initial folding of the pro-alpha chain in the endoplasmic reticulum, resulting in its retention and degradation. A much less common occurrence is a mutation that specifically affects an "active-site" residue necessary for substrate binding and/or catalysis. In this case, hexosaminidase A is present in the lysosome, but it lacks all alpha-specific activity. This biochemical phenotype is referred to as the "B1-variant form" of Tay-Sachs disease. Kinetic analysis of suspected B1-variant mutations is complex because hexosaminidase A is heterodimeric and both subunits possess similar active sites. In this report, we examine a previously identified B1-variant mutation, alpha-Val192Leu. Chinese hamster ovary cells were permanently cotransfected with an alpha-cDNA-construct encoding the substitution and a mutant beta-cDNA (beta-Arg211Lys), encoding a beta-subunit that is inactive but normal in all other respects. We were surprised to find that the Val192Leu substitution, produced a pro-alpha chain that did not form alpha-beta dimers and was not transported to the lysosome. Finally, we reexamined the hexosaminidase activity and protein levels in the fibroblasts from the original patient. These data were also not consistent with the biochemical phenotype of the B1 variant of Tay-Sachs disease previously reported to be present. Thus, we conclude that the Val192Leu substitution does not specifically affect the alpha-active site.

Satellite-tracking and earth-dynamics research programs. [NASA Programs on satellite orbits and satellite ground tracks of geodetic satellites

NASA Technical Reports Server (NTRS)

1974-01-01

Observations and research progress of the Smithsonian Astrophysical Observatory are reported. Satellite tracking networks (ground stations) are discussed and equipment (Baker-Nunn cameras) used to observe the satellites is described. The improvement of the accuracy of a laser ranging system of the ground stations is discussed. Also, research efforts in satellite geodesy (tides, gravity anomalies, plate tectonics) is discussed. The use of data processing for geophysical data is examined, and a data base for the Earth and Ocean Physics Applications Program is proposed. Analytical models of the earth's motion (computerized simulation) are described and the computation (numerical integration and algorithms) of satellite orbits affected by the earth's albedo, using computer techniques, is also considered. Research efforts in the study of the atmosphere are examined (the effect of drag on satellite motion), and models of the atmosphere based on satellite data are described.

A unique chromatin complex occupies young α-satellite arrays of human centromeres

PubMed Central

Henikoff, Jorja G.; Thakur, Jitendra; Kasinathan, Sivakanthan; Henikoff, Steven

2015-01-01

The intractability of homogeneous α-satellite arrays has impeded understanding of human centromeres. Artificial centromeres are produced from higher-order repeats (HORs) present at centromere edges, although the exact sequences and chromatin conformations of centromere cores remain unknown. We use high-resolution chromatin immunoprecipitation (ChIP) of centromere components followed by clustering of sequence data as an unbiased approach to identify functional centromere sequences. We find that specific dimeric α-satellite units shared by multiple individuals dominate functional human centromeres. We identify two recently homogenized α-satellite dimers that are occupied by precisely positioned CENP-A (cenH3) nucleosomes with two ~100–base pair (bp) DNA wraps in tandem separated by a CENP-B/CENP-C–containing linker, whereas pericentromeric HORs show diffuse positioning. Precise positioning is largely maintained, whereas abundance decreases exponentially with divergence, which suggests that young α-satellite dimers with paired ~100-bp particles mediate evolution of functional human centromeres. Our unbiased strategy for identifying functional centromeric sequences should be generally applicable to tandem repeat arrays that dominate the centromeres of most eukaryotes. PMID:25927077

Secretion of alpha 2-plasmin inhibitor is impaired by amino acid deletion in a small region of the molecule.

PubMed

Toyota, S; Hirosawa, S; Aoki, N

1994-02-01

Alpha 2-plasmin inhibitor (alpha 2PI) deficiency Okinawa results from defective secretion of the inhibitor from the liver and appears to be a direct consequence of the deletion of Glu137 in the amino acid sequence of alpha 2PI. To examine the effects of replacing the amino acid occupying position 137 or deleting its neighboring amino acid on alpha 2PI secretion, we used oligonucleotide-directed mutagenesis of alpha 2PI cDNA to change the codon specifying Glu137 or delete a codon specifying its neighboring amino acid. The effects were determined by pulse-chase experiments and by enzyme-linked immunosorbent assay of media from transiently transfected COS-7 cells. Replacement of Glu137 with an amino acid other than Cys had little effect on alpha 2PI secretion. In contrast, deletion of an amino acid in a region spanning a sequence of less than 30 amino acids including positions 127 and 137 severely impaired the secretion. The results suggest that structural integrity of the region, rather than its component amino acids, is important for the intracellular transport and secretion of alpha 2PI.

Mapping High-Velocity H-alpha and Lyman-alpha Emission from Supernova 1987A

NASA Technical Reports Server (NTRS)

France, Kevin; McCray, Richard; Fransson, Claes; Larsson, Josefin; Frank, Kari A.; Burrows, David N.; Challis, Peter; Kirshner, Robert P.; Chevalier, Roger A.; Garnavich, Peter;

Morphological characterization of the AlphaA- and AlphaB-crystallin double knockout mouse lens

PubMed Central

Boyle, Daniel L; Takemoto, Larry; Brady, James P; Wawrousek, Eric F

2003-01-01

Background One approach to resolving some of the in vivo functions of alpha-crystallin is to generate animal models where one or both of the alpha-crystallin gene products have been eliminated. In the single alpha-crystallin knockout mice, the remaining alpha-crystallin may fully or partially compensate for some of the functions of the missing protein, especially in the lens, where both alphaA and alphaB are normally expressed at high levels. The purpose of this study was to characterize gross lenticular morphology in normal mice and mice with the targeted disruption of alphaA- and alphaB-crystallin genes (alphaA/BKO). Methods Lenses from 129SvEvTac mice and alphaA/BKO mice were examined by standard scanning electron microscopy and confocal microscopy methodologies. Results Equatorial and axial (sagittal) dimensions of lenses for alphaA/BKO mice were significantly smaller than age-matched wild type lenses. No posterior sutures or fiber cells extending to the posterior capsule of the lens were found in alphaA/BKO lenses. Ectopical nucleic acid staining was observed in the posterior subcapsular region of 5 wk and anterior subcapsular cortex of 54 wk alphaA/BKO lenses. Gross morphological differences were also observed in the equatorial/bow, posterior and anterior regions of lenses from alphaA/BKO mice as compared to wild mice. Conclusion These results indicated that both alphaA- and alphaB-crystallin are necessary for proper fiber cell formation, and that the absence of alpha-crystallin can lead to cataract formation. PMID:12546709

DNA-based identification and phylogeny of North American Armillaria species

Treesearch

Amy L. Ross-Davis; John W. Hanna; Ned B. Klopfenstein

2011-01-01

Because Armillaria species display different ecological behaviors across diverse forest ecosystems, it is critical to identify Armillaria species accurately for any assessment of forest health. To further develop DNA-based identification methods, partial sequences of the translation elongation factor-1 alpha (EF-1α) gene were used to examine the phylogenetic...

Cell-extracellular matrix interactions can regulate the switch between growth and differentiation in rat hepatocytes: reciprocal expression of C/EBP alpha and immediate-early growth response transcription factors.

PubMed Central

Rana, B; Mischoulon, D; Xie, Y; Bucher, N L; Farmer, S R

1994-01-01

Previous investigations have shown that culture of freshly isolated hepatocytes under conventional conditions, i.e., on dried rat tail collagen in the presence of growth factors, facilitates cell growth but also causes an extensive down-regulation of most liver-specific functions. This dedifferentiation process can be prevented if the cells are cultured on a reconstituted basement membrane gel matrix derived from the Englebreth-Holm-Swarm mouse sarcoma tumor (EHS gel). To gain insight into the mechanisms regulating this response to extracellular matrix, we are analyzing the activities of two families of transcription factors, C/EBP and AP-1, which control the transcription of hepatic and growth-responsive genes, respectively. We demonstrate that isolation of hepatocytes from the normal quiescent rat liver by collagenase perfusion activates the immediate-early growth response program, as indicated by increased expression of c-jun, junB, c-fos, and c-myc mRNAs. Adhesion of these activated cells to dried rat tail collagen augments the elevated levels of these mRNAs for the initial 1 to 2 h postplating; junB and c-myc mRNA levels then drop steeply, with junB returning to normal quiescence and the c-myc level remaining slightly elevated during the 3-day culture period. Levels of c-jun mRNA and AP-1 DNA binding activity, however, remain elevated from the outset, while C/EBP alpha mRNA expression is down-regulated, resulting in a decrease in the steady-state levels of the 42- and 30-kDa C/EBP alpha polypeptides and C/EBP alpha DNA binding activity. In contrast, C/EBP beta mRNA production remains at near-normal hepatic levels for 5 to 8 days of culture, although its DNA binding activity decreases severalfold during this time. Adhesion of hepatocytes to the EHS gel for the same period of time dramatically alters this program: it arrests growth and inhibits AP-1 DNA binding activity and the expression of c-jun, junB, and c-myc mRNAs, but, in addition, it restores C/EBP alpha

A defective retroviral vector encoding human interferon-alpha2 can transduce human leukemic cell lines.

PubMed

Austruy, E; Bagnis, C; Carbuccia, N; Maroc, C; Birg, F; Dubreuil, P; Mannoni, P; Chabannon, C

1998-01-01

Using the LXSN backbone, a defective retroviral vector (LISN) was constructed that encodes the human interferon (IFN)-alpha2 (hIFN-alpha2) gene and the neomycin resistance gene; the hIFN-alpha2 gene was cloned from human placental genomic DNA. High titers of the LISN retrovirus were produced by the amphotropic packaging cell line GP+envAM12. LISN is able to infect three human hematopoietic and leukemic cell lines: K562, LAMA-84, and TF-1. G418-resistant cells were detected in a similar proportion after infection with either the LISN retroviral vector or the LnLSN retroviral vector (encoding the nlsLacZ gene instead of hIFN-alpha2), suggesting that hIFN-alpha2 does not inhibit (or only partially inhibits) the production of retroviral particles by the packaging cell line and the infection of human cells. LISN-infected cells express and secrete hIFN-alpha2 as demonstrated by Northern blot analysis of poly(A)+ RNA, detection of the intracellular protein by fluorescence-activated cell sorter analysis, and detection of secreted hIFN-alpha in cell supernatants using an enzyme-linked immunosorbent assay. Retrovirally produced hIFN-alpha2 is biologically active, as demonstrated by the partial inhibition of the growth of K562 and TF-1, the modulation of the expression of cell surface antigens, the induction of the (2'-5') oligoadenylate synthetase, and, for LAMA-84, the down-modulation of the BCR-ABL protein. We conclude that the infection of human leukemic cell lines with a retroviral vector encoding hIFN-alpha2 is feasible and induces the expected biological effects. This experimental model will be useful in investigating the possibility of transducing normal and leukemic cells and hematopoietic progenitors and in determining the consequences of the autocrine production of hIFN-alpha2 on the behavior of these cells.

Solution conformation of a neuronal nicotinic acetylcholine receptor antagonist {alpha}-conotoxin OmIA that discriminates {alpha}3 vs. {alpha}6 nAChR subtypes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chi, Seung-Wook; Kim, Do-Hyoung; Olivera, Baldomero M.

2006-06-23

{alpha}-Conotoxin OmIA from Conus omaria is the only {alpha}-conotoxin that shows a {approx}20-fold higher affinity to the {alpha}3{beta}2 over the {alpha}6{beta}2 subtype of nicotinic acetylcholine receptor. We have determined a three-dimensional structure of {alpha}-conotoxin OmIA by nuclear magnetic resonance spectroscopy. {alpha}-Conotoxin OmIA has an '{omega}-shaped' overall topology with His{sup 5}-Asn{sup 12} forming an {alpha}-helix. Structural features of {alpha}-conotoxin OmIA responsible for its selectivity are suggested by comparing its surface characteristics with other functionally related {alpha}4/7 subfamily conotoxins. Reduced size of the hydrophilic area in {alpha}-conotoxin OmIA seems to be associated with the reduced affinity towards the {alpha}6{beta}2 nAChR subtype.

Proline-catalysed asymmetric amination of alpha,alpha-disubstituted aldehydes: synthesis of configurationally stable enantioenriched alpha-aminoaldehydes.

PubMed

Vogt, Henning; Vanderheiden, Sylvia; Bräse, Stefan

2003-10-07

Proline-catalysed amination of alpha,alpha-disubstituted racemic aldehydes with azodicarboxylates proceeds smoothly to give configurationally stable scalemic aldehydes and oxazolidinones in up to 86% ee.

Hepatic global DNA and peroxisome proliferator-activated receptor alpha promoter methylation are altered in peripartal dairy cows fed rumen-protected methionine.

PubMed

Osorio, J S; Jacometo, C B; Zhou, Z; Luchini, D; Cardoso, F C; Loor, J J

2016-01-01

The availability of Met in metabolizable protein (MP) of a wide range of diets for dairy cows is low. During late pregnancy and early lactation, in particular, suboptimal Met in MP limits its use for mammary and liver metabolism and also for the synthesis of S-adenosylmethionine, which is essential for many biological processes, including DNA methylation. The latter is an epigenetic modification involved in the regulation of gene expression, hence, tissue function. Thirty-nine Holstein cows were fed throughout the peripartal period (-21 d to 30 d in milk) a basal control (CON) diet (n=14) with no Met supplementation, CON plus MetaSmart (MS; Adisseo NA, Alpharetta, GA; n=12), or CON plus Smartamine M (SM; Adisseo NA; n=13). The total mixed ration dry matter for the close-up and lactation diets was measured weekly, then the Met supplements were adjusted daily and top-dressed over the total mixed ration at a rate of 0.19 (MS) or 0.07% (SM) on a dry matter basis. Liver tissue was collected on -10, 7, and 21 d for global DNA and peroxisome proliferator-activated receptor alpha (PPARα) promoter region-specific methylation. Several PPARα target and putative target genes associated with carnitine synthesis and uptake, fatty acid metabolism, hepatokines, and carbohydrate metabolism were also studied. Data were analyzed using PROC MIXED of SAS (SAS Institute Inc., Cary, NC) with the preplanned contrast CON versus SM + MS. Global hepatic DNA methylation on d 21 postpartum was lower in Met-supplemented cows than CON. However, of 2 primers used encompassing 4 to 12 CpG sites in the promoter region of bovine PPARA, greater methylation occurred in the region encompassing -1,538 to -1,418 from the transcription start site in cows supplemented with Met. Overall expression of PPARA was greater in Met-supplemented cows than CON. Concomitantly, PPARA-target genes, such as ANGPTL4, FGF21, and PCK1, were also upregulated overall by Met supplementation. The upregulation of PPAR�

Antisense suppression of phospholipase D alpha retards abscisic acid- and ethylene-promoted senescence of postharvest Arabidopsis leaves.

PubMed Central

Fan, L; Zheng, S; Wang, X

1997-01-01

Membrane disruption has been proposed to be a key event in plant senescence, and phospholipase D (PLD; EC 3.1.4.4) has been thought to play an important role in membrane deterioration. We recently cloned and biochemically characterized three different PLDs from Arabidopsis. In this study, we investigated the role of the most prevalent phospholipid-hydrolyzing enzyme, PLD alpha, in membrane degradation and senescence in Arabidopsis. The expression of PLD alpha was suppressed by introducing a PLD alpha antisense cDNA fragment into Arabidopsis. When incubated with abscisic acid and ethylene, leaves detached from the PLD alpha-deficient transgenic plants showed a slower rate of senescence than did those from wild-type and transgenic control plants. The retardation of senescence was demonstrated by delayed leaf yellowing, lower ion leakage, greater photosynthetic activity, and higher content of chlorophyll and phospholipids in the PLD alpha antisense leaves than in those of the wild type. Treatment of detached leaves with abscisic acid and ethylene stimulated PLD alpha expression, as indicated by increases in PLD alpha mRNA, protein, and activity. In the absence of abscisic acid and ethylene, however, detached leaves from the PLD alpha-deficient and wild-type plants showed a similar rate of senescence. In addition, the suppression of PLD alpha did not alter natural plant growth and development. These data suggest that PLD alpha is an important mediator in phytohormone-promoted senescence in detached leaves but is not a direct promoter of natural senescence. The physiological relevance of these findings is discussed. PMID:9437863

Discrimination of Single Base Pair Differences Among Individual DNA Molecules Using a Nanopore

NASA Technical Reports Server (NTRS)

Vercoutere, Wenonah; DeGuzman, Veronica

2003-01-01

The protein toxin alpha-hemolysin form nanometer scale channels across lipid membranes. Our lab uses a single channel in an artificial lipid bilayer in a patch clamp device to capture and examine individual DNA molecules. This nanopore detector used with a support vector machine (SVM) can analyze DNA hairpin molecules on the millisecond time scale. We distinguish duplex stem length, base pair mismatches, loop length, and single base pair differences. The residual current fluxes also reveal structural molecular dynamics elements. DNA end-fraying (terminal base pair dissociation) can be observed as near full blockades, or spikes, in current. This technique can be used to investigate other biological processes dependent on DNA end-fraying, such as the processing of HIV DNA by HIV integrase.