scholarly journals Characterization of the manganese superoxide dismutase cDNA and gene from the human parasite Onchocerca volvulus

1995 ◽  
Vol 308 (2) ◽  
pp. 441-446 ◽  
Author(s):  
K Henkle-Dührsen ◽  
W Tawe ◽  
C Warnecke ◽  
R D Walter
Keyword(s):  
Superoxide Dismutase ◽  
Nucleotide Sequence ◽  
Adult Worm ◽  
Genomic Dna ◽  
Genomic Library ◽  
Regulatory Elements ◽  
Onchocerca Volvulus ◽  
Flanking Sequence ◽  
Degenerate Primers ◽  
Total Rna

The manganese-containing superoxide dismutase (MnSOD) is a major component of the cellular defence mechanisms against the toxic effects of the superoxide radical. Within the framework of studies on anti-oxidant enzymes and their protective role in the human parasitic nematode Onchocerca volvulus, sequences encoding the MnSOD were isolated and examined in this study. Degenerate primers were designed based upon conserved regions of MnSOD sequences from other organisms, and were used in PCR on reverse-transcribed O. volvulus total RNA and genomic DNA to identify partial cDNA and genomic DNA fragments encoding the O. volvulus MnSOD (OvMnSOD). The genomic DNA PCR product was used to screen an O. volvulus adult worm lambda unizap II cDNA library and the nucleotide sequence of the longest clone determined. The complete 5′-end of the OvMnSOD cDNA was obtained using the rapid amplification of cDNA ends (RACE) procedure with O. volvulus total RNA and was found to possess a spliced leader sequence at the 5′-terminus. The deduced primary sequence encodes a 25 kDa protein, which has the conserved residues required for enzyme activity and metal binding. The 24 N-terminal amino acids encoded by the OvMnSOD cDNA comprise a putative mitochondrial transit peptide. The OvMnSOD gene was also isolated from an O. volvulus adult worm lambda fix II genomic library, a restriction map was constructed and the nucleotide sequence determined. The OvMnSOD gene was found to possess five exons and four introns with consensus splice-site junctions. Potential regulatory elements were identified in the 5′ genomic flanking sequence. Southern-blot analysis with total worm genomic DNA indicates a single-copy gene, with a restriction pattern consistent with that of the isolated gene.

scholarly journals Characterization of an Atypical Superoxide Dismutase from Sinorhizobium meliloti

1999 ◽  
Vol 181 (15) ◽  
pp. 4509-4516 ◽  
Author(s):  
Renata Santos ◽  
Stephane Bocquet ◽  
Alain Puppo ◽  
Danièle Touati
Keyword(s):  
Superoxide Dismutase ◽  
Amino Acid ◽  
Sinorhizobium Meliloti ◽  
Genomic Library ◽  
Specific Activity ◽  
Growth Conditions ◽  
Aerobic Bacterium ◽  
Degenerate Primers ◽  
E Coli ◽  
Acid Protein

ABSTRACT Sinorhizobium meliloti Rm5000 is an aerobic bacterium that can live free in the soil or in symbiosis with the roots of leguminous plants. A single detectable superoxide dismutase (SOD) was found in free-living growth conditions. The corresponding gene was isolated from a genomic library by using a sod fragment amplified by PCR from degenerate primers as a probe. ThesodA gene was located in the chromosome. It is transcribed monocistronically and encodes a 200-amino-acid protein with a theoretical M r of 22,430 and pI of 5.8.S. meliloti SOD complemented a deficient E. coli mutant, restoring aerobic growth of a sodA sodB recA strain, when the gene was expressed from the synthetictac promoter but not from its own promoter. Amino acid sequence alignment showed great similarity with Fe-containing SODs (FeSODs), but the enzyme was not inactivated by H2O2. The native enzyme was purified and found to be a dimeric protein, with a specific activity of 4,000 U/mg. Despite its Fe-type sequence, atomic absorption spectroscopy showed manganese to be the cofactor (0.75 mol of manganese and 0.24 mol of iron per mol of monomer). The apoenzyme was prepared from crude extracts of S. meliloti. Activity was restored by dialysis against either MnCl2 or Fe(NH4)2(SO4)2, demonstrating the cambialistic nature of the S. melilotiSOD. The recovered activity with manganese was sevenfold higher than with iron. Both reconstituted enzymes were resistant to H2O2. Sequence comparison with 70 FeSODs and MnSODs indicates that S. meliloti SOD contains several atypical residues at specific sites that might account for the activation by manganese and resistance to H2O2of this unusual Fe-type SOD.

scholarly journals Evidence for Positive and Negative Regulatory Elements in the 5′-Flanking Sequence of the Mouse Sparc (osteonectin) Gene

1989 ◽  
Vol 264 (21) ◽  
pp. 12201-12207 ◽  
Author(s):  
S Nomura ◽  
S Hashmi ◽  
J H McVey ◽  
J Ham ◽  
M Parker ◽  
...  
Keyword(s):  
Regulatory Elements ◽  
Flanking Sequence

scholarly journals Chicken beta B1-crystallin gene expression: presence of conserved functional polyomavirus enhancer-like and octamer binding-like promoter elements found in non-lens genes.

1991 ◽  
Vol 11 (3) ◽  
pp. 1488-1499 ◽  
Author(s):  
H J Roth ◽  
G C Das ◽  
J Piatigorsky
Keyword(s):  
Transcription Factors ◽  
Hela Cell ◽  
Dna Sequences ◽  
Nuclear Extract ◽  
Regulatory Elements ◽  
Dnase I ◽  
Flanking Sequence ◽  
Mobility Shift ◽  
Promoter Elements ◽  
Dnase I Footprinting

Expression of the chicken beta B1-crystallin gene was examined. Northern (RNA) blot and primer extension analyses showed that while abundant in the lens, the beta B1 mRNA is absent from the liver, brain, heart, skeletal muscle, and fibroblasts of the chicken embryo, suggesting lens specificity. Promoter fragments ranging from 434 to 126 bp of 5'-flanking sequence (plus 30 bp of exon 1) of the beta B1 gene fused to the bacterial chloramphenicol acetyltransferase gene functioned much more efficiently in transfected embryonic chicken lens epithelial cells than in transfected primary muscle fibroblasts or HeLa cells. Transient expression of recombinant plasmids in cultured lens cells, DNase I footprinting, in vitro transcription in a HeLa cell extract, and gel mobility shift assays were used to identify putative functional promoter elements of the beta B1-crystallin gene. Sequence analysis revealed a number of potential regulatory elements between positions -126 and -53 of the beta B1 promoter, including two Sp1 sites, two octamer binding sequence-like sites (OL-1 and OL-2), and two polyomavirus enhancer-like sites (PL-1 and PL-2). Deletion and site-specific mutation experiments established the functional importance of PL-1 (-116 to -102), PL-2 (-90 to -76), and OL-2 (-75 to -68). DNase I footprinting using a lens or a HeLa cell nuclear extract and gel mobility shifts using a lens nuclear extract indicated the presence of putative lens transcription factors binding to these DNA sequences. Competition experiments provided evidence that PL-1 and PL-2 recognize the same or very similar factors, while OL-2 recognizes a different factor. Our data suggest that the same or closely related transcription factors found in many tissues are used for expression of the chicken beta B1-crystallin gene in the lens.

scholarly journals First Report of Mosaic Disease Caused by Colombian datura virus on Solanum lycopersicum Plants Commercially Cultivated in Japan

Plant Disease ◽  
2014 ◽  
Vol 98 (5) ◽  
pp. 698-698 ◽  
Author(s):  
Y. Tomitaka ◽  
T. Usugi ◽  
R. Kozuka ◽  
S. Tsuda
Keyword(s):  
Nucleotide Sequence ◽  
Solanum Lycopersicum ◽  
Mosaic Disease ◽  
Causal Agent ◽  
Cultivated Plants ◽  
Rt Pcr ◽  
Degenerate Primers ◽  
Specific Primers ◽  
Tomato Plants ◽  
First Report

In 2009, some commercially grown tomato (Solanum lycopersicum) plants in Chiba Prefecture, Japan, exhibited mosaic symptoms. Ten plants from a total of about 72,000 cultivated plants in the greenhouses showed such symptoms. To identify the causal agent, sap from leaves of the diseased plants was inoculated into Chenopodium quinoa and Nicotiana benthamiana plants. Local necrotic lesions appeared on inoculated leaves of C. quinoa, but no systemic infection was observed. Systemic mosaic symptoms were observed on the N. benthamiana plants inoculated. Single local lesion isolation was performed three times using C. quinoa to obtain a reference isolate for further characterization. N. benthamiana was used for propagation of the isolate. Sap from infected leaves of N. benthamiana was mechanically inoculated into three individual S. lycopersicum cv. Momotaro. Symptoms appearing on inoculated tomatoes were indistinguishable from those of diseased tomato plants found initially in the greenhouse. Flexuous, filamentous particles, ~750 nm long, were observed by electron microscopy in the sap of the tomato plants inoculated with the isolate, indicating that the infecting virus may belong to the family Potyviridae. To determine genomic sequence of the virus, RT-PCR was performed. Total RNA was extracted from the tomato leaves experimentally infected with the isolate using an RNeasy Plant Mini kit (QIAGEN, Hilden, Germany). RT-PCR was performed by using a set of universal, degenerate primers for Potyviruses as previously reported (2). Amplicons (~1,500 bp) generated by RT-PCR were extracted from the gels using the QIAquick Gel Extraction kit (QIAGEN) and cloned into pCR-BluntII TOPO (Invitrogen, San Diego, CA). DNA sequences of three individual clones were determined using a combination of plasmid and virus-specific primers, showing that identity among three clones was 99.8%. A consensus nucleotide sequence of the isolate was deposited in GenBank (AB823816). BLASTn analysis of the nucleotide sequence determined showed 99% identity with a partial sequence in the NIb/coat protein (CP) region of Colombian datura virus (CDV) tobacco isolate (JQ801448). Comparison of the amino acid sequence predicted for the CP with previously reported sequences for CDV (AY621656, AJ237923, EU571230, AM113759, AM113754, and AM113761) showed 97 to 100% identity range. Subsequently, CDV infection in both the original and experimentally inoculated plants was confirmed by RT-PCR using CDV-specific primers (CDVv and CDVvc; [1]), and, hence, the causal agent of the tomato disease observed in greenhouse tomatoes was proved to be CDV. The first case of CDV on tomato was reported in Netherlands (3), indicating that CDV was transmitted by aphids from CDV-infected Brugmansia plants cultivated in the same greenhouse. We carefully investigated whether Brugmansia plants naturally grew around the greenhouses, but we could not find them inside or in proximity to the greenhouses. Therefore, sources of CDV inoculum in Japan are still unclear. This is the first report of a mosaic disease caused by CDV on commercially cultivated S. lycopersicum in Japan. References: (1) D. O. Chellemi et al. Plant Dis. 95:755, 2011. (2) J. Chen et al. Arch. Virol. 146:757, 2001. (3) J. Th. J. Verhoeven et al. Eur. J. Plant. Pathol. 102:895, 1996.

Novel tenascin variants with a distinctive pattern of expression in the avian embryo

Development ◽  
1994 ◽  
Vol 120 (3) ◽  
pp. 637-647
Author(s):  
R.P. Tucker ◽  
J. Spring ◽  
S. Baumgartner ◽  
D. Martin ◽  
C. Hagios ◽  
...  
Keyword(s):  
Genomic Library ◽  
Chicken Embryo Fibroblast ◽  
Cdna Probe ◽  
Avian Embryo ◽  
Degenerate Primers ◽  
Type Iii ◽  
Embryonic Tissues ◽  
Fibronectin Type Iii ◽  
Fibronectin Type

Previous studies have shown that several forms of the glycoprotein tenascin are present in the embryonic extracellular matrix. These forms are the result of alternative splicing, which generates tenascin variants with different numbers of fibronectin type III repeats. We have used degenerate primers and PCR to isolate a novel tenascin exon from an avian genomic library. Genomic clones contained a sequence encoding a fibronectin type III repeat that corresponds to repeat ‘C’ from the variable domain of human tenascin. To demonstrate that tenascin containing repeat ‘C’ is actually synthesized by avian cells, a monospecific antiserum was raised against a repeat ‘C’ fusion protein. This antiserum recognized a novel high-molecular-weight variant on immunoblots of tenascin isolated from chicken embryo fibroblast-conditioned medium, and stained tendons on frozen sections of chicken embryos. A cDNA probe specific for mRNA encoding repeat ‘C’ was used for in situ hybridization. This probe hybridized in a subset of the embryonic tissues labelled with a universal tenascin probe, including tendons, ligaments and mesenchyme at sites of epithelial-mesenchymal interactions. Finally, we provide evidence that additional fibronectin type III repeats, one corresponding to a recently discovered human repeat as well as one entirely novel sequence, also exists in chicken tenascin mRNA. These data indicate that tenascin is present in the embryonic matrix in a multitude of forms and that these forms have distinctive distributions that may reflect more than one function for tenascin in development.

scholarly journals Identification of Streptococci to Species Level by Sequencing the Gene Encoding the Manganese-Dependent Superoxide Dismutase

1998 ◽  
Vol 36 (1) ◽  
pp. 41-47 ◽  
Author(s):  
Claire Poyart ◽  
Gilles Quesne ◽  
Stephane Coulon ◽  
Patrick Berche ◽  
Patrick Trieu-Cuot
Keyword(s):  
Superoxide Dismutase ◽  
Pcr Assay ◽  
Degenerate Primers ◽  
Gene Encoding ◽  
Genes Encoding ◽  
Type Strains ◽  
Internal Fragment ◽  
Rrna Sequences ◽  
16S Rrna Sequences

We have used a PCR assay based on the use of degenerate primers in order to characterize an internal fragment (sodAint ) representing approximately 85% of the genes encoding the manganese-dependent superoxide dismutase in various streptococcal type strains (S. acidominimus,S. agalactiae, S. alactolyticus, S. anginosus, S. bovis, S. constellatus,S. canis, S. cricetus, S. downei,S. dysgalactiae, S. equi subsp.equi, S. equi subsp. zooepidemicus,S. equinus, S. gordonii, S. iniae,S. intermedius, S. mitis, S. mutans, S. oralis, S. parasanguis,S. pneumoniae, S. porcinus, S. pyogenes, S. salivarius, S. sanguis,S. sobrinus, S. suis, S. thermophilus, and S. vestibularis). Phylogenetic analysis of these sodAint fragments yields an evolutionary tree having a topology similar to that of the tree constructed with the 16S rRNA sequences. We have shown that clinical isolates could be identified by determining the positions of theirsodAint fragments on the phylogenetic tree of the sodAint fragments of the type species. We propose this method for the characterization of strains that cannot be assigned to a species on the basis of their conventional phenotypic reactions.

Molecular and cytogenetic analysis of repetitive DNA in pea (Pisum sativum L.)

Genome ◽  
2001 ◽  
Vol 44 (4) ◽  
pp. 716-728 ◽  
Author(s):  
Pavel Neumann ◽  
Marcela Nouzová ◽  
Jirí Macas
Keyword(s):  
Pisum Sativum ◽  
Repetitive Dna ◽  
Dna Sequences ◽  
Genomic Dna ◽  
Tandem Repeats ◽  
Genomic Library ◽  
Chromosome Morphology ◽  
Plant Genome ◽  
Genomic Repeats ◽  
Dispersed Repeats

A set of pea DNA sequences representing the most abundant genomic repeats was obtained by combining several approaches. Dispersed repeats were isolated by screening a short-insert genomic library using genomic DNA as a probe. Thirty-two clones ranging from 149 to 2961 bp in size and from 1000 to 39 000/1C in their copy number were sequenced and further characterized. Fourteen clones were identified as retrotransposon-like sequences, based on their homologies to known elements. Fluorescence in situ hybridization using clones of reverse transcriptase and integrase coding sequences as probes revealed that corresponding retroelements were scattered along all pea chromosomes. Two novel families of tandem repeats, named PisTR-A and PisTR-B, were isolated by screening a genomic DNA library with Cot-1 DNA and by employing genomic self-priming PCR, respectively. PisTR-A repeats are 211–212 bp long, their abundance is 2 × 104 copies/1C, and they are partially clustered in a secondary constriction of one chromosome pair with the rest of their copies dispersed on all chromosomes. PisTR-B sequences are of similar abundance (104 copies/1C) but differ from the "A" family in their monomer length (50 bp), high A/T content, and chromosomal localization in a limited number of discrete bands. These bands are located mainly in (sub)telomeric and pericentromeric regions, and their patterns, together with chromosome morphology, allow discrimination of all chromosome types within the pea karyotype. Whereas both tandem repeat families are mostly specific to the genus Pisum, many of the dispersed repeats were detected in other legume species, mainly those in the genus Vicia.Key words: repetitive DNA, plant genome, retroelements, satellite DNA, Pisum sativum.

Predicted structure of the bovine calcitonin gene-related peptide and the carboxy-terminal flanking peptide of bovine calcitonin precursor

1991 ◽  
Vol 6 (2) ◽  
pp. 147-152 ◽  
Author(s):  
K. Collyear ◽  
S. I. Girgis ◽  
G. Saunders ◽  
I. MacIntyre ◽  
G. Holt
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Nucleotide Sequence ◽  
Genomic Library ◽  
Amino Acid Peptide ◽  
Related Peptide ◽  
Calcitonin Gene ◽  
Significant Homology ◽  
Human Counterpart ◽  
Carboxy Terminal

ABSTRACT We have isolated from a bovine genomic library a clone which contains the calcitonin (CT) and CT gene-related peptide (CGRP) sequences, using probes representing the human CT and CGRP sequences. Sequence analysis has identified the nucleotide sequence coding for bovine CT, its C-terminal flanking peptide and bovine CGRP. The deduced amino acid sequence of bovine CGRP revealed a significant homology with other CGRPs so far reported. It differs by only one amino acid from rat CGRPα and porcine CGRP, and by three and four amino acids from human CGRPβ and α respectively. Bovine CT has, however, only 14 out of 32 residues in common with human CT. As in the human CT precursor, the C-terminal flanking peptide of bovine CT precursor is a 21 amino acid peptide. It shares only 11 residues in common with its human counterpart. This study thus provides further evidence that CGRP, in contrast to CT and its C-terminal flanking peptide, is a highly conserved molecule.

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