scholarly journals Sequencing, Improved Detection, and a Novel Form of Kalanchoë top-spotting virus

Plant Disease ◽  
2005 ◽  
Vol 89 (3) ◽  
pp. 298-302 ◽  
Author(s):  
Zihong Yang ◽  
Mogens Nicolaisen ◽  
Neil E. Olszewski ◽  
B. E. L. Lockhart
Keyword(s):  
Genomic Sequence ◽  
Pcr Amplification ◽  
Leaf Tissue ◽  
Open Reading Frames ◽  
Oligonucleotide Primer ◽  
Infected Leaf ◽  
Breeding Lines ◽  
Double Stranded Dna ◽  
Extraction Step ◽  
Polymerase Chain

Virions of Kalanchoë top-spotting virus (KTSV) were purified from infected leaf tissue of Kalanchoë blossfeldiana using a procedure that prevented loss of virus in the initial extraction step. The double-stranded DNA viral genome was cloned and sequenced. The KTSV genome was 7,591 bp in size and contained three open reading frames capable of encoding proteins of 21, 14, and 223 kDa, respectively. The size and organization of the KTSV genome were similar to those of other mealybug-transmitted badnaviruses. Several oligonucleotide primer pairs, based on the KTSV genomic sequence, were used to efficiently detect the virus in plants, thereby removing a major constraint to reliable screening of kalanchoë propagating stock and breeding lines for KTSV infection. Two KTSV sequences, one symptom-inducing and the other not, were identified and differentiated by polymerase chain reaction (PCR) amplification and digestion of the resulting amplicon with restriction endonucleases. Preliminary results from graft-transmission tests and PCR indexing suggest that the nonsymptomatic form of KTSV may represent an integrated viral element. The occurrence of such integrated pararetroviral elements poses practical problems for routine PCR indexing of breeding and propagating stock, and also raises the possibility of symptomatic episomal infections arising from these viral integrants.

scholarly journals Complete Genome Sequence of Salmonella Bacteriophage SS3e

2012 ◽  
Vol 86 (18) ◽  
pp. 10253-10254 ◽  
Author(s):  
Sung-Hun Kim ◽  
Jeong-Hyun Park ◽  
Bok-Kwon Lee ◽  
Hyuk-Joon Kwon ◽  
Ji-Hyun Shin ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Sequence Analysis ◽  
Genomic Sequence ◽  
Enterobacter Cloacae ◽  
Open Reading Frames ◽  
Lytic Bacteriophage ◽  
Content Type ◽  
Double Stranded Dna ◽  
Genomic Sequence Analysis ◽  
Reading Frames

ASalmonellalytic bacteriophage, SS3e, was isolated, and its genome was sequenced completely. This phage is able to lyse not only variousSalmonellaserovars but alsoEscherichia coli,Shigella sonnei,Enterobacter cloacae, andSerratia marcescens, indicating a broad host specificity. Genomic sequence analysis of SS3e revealed a linear double-stranded DNA sequence of 40,793 bp harboring 58 open reading frames, which is highly similar toSalmonellaphages SETP13 and MB78.

scholarly journals Complete Genome Sequence of a Putative Novel Ilarvirus Isolated From Eleocharis Dulcis

2021 ◽  
Author(s):  
Lanqing Lv ◽  
Xinyang Wu ◽  
Jiajia Weng ◽  
Yuchao Lai ◽  
Kelei Han ◽  
...  
Keyword(s):  
Genomic Sequence ◽  
Phylogenetic Analyses ◽  
Genetic Distances ◽  
Open Reading Frames ◽  
Serological Reaction ◽  
Water Chestnut ◽  
Polymerase Chain ◽  
Rapid Amplification ◽  
Race Pcr ◽  
Next Generation Sequencing Ngs

Abstract The complete genomic sequence of a novel ilarvirus from Eleocharis dulcis, tentatively named water chestnut virus A (WCVA), was determined using next generation sequencing (NGS) combined with reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) PCR. The three genomic RNA components of WCVA were 3578 (RNA1), 2873 (RNA2) and 2073 (RNA3) nucleotides long, with four predicted open reading frames containing conserved domains and motifs typical of ilarviruses. Phylogenetic analyses of each predicted protein consistently placed WCVA in subgroup 4 of the genus Ilarvirus, together with prune dwarf virus, viola white distortion associated virus, fragaria chiloensis latent virus and potato yellowing virus. The genetic distances and lack of serological reaction to antisera of other ilarviruses suggest that WCVA is a novel member of the genus.

scholarly journals The Previously Unidentified, Divergent Badnavirus Species Cacao red vein-banding virus is Associated with Cacao Swollen Shoot Disease in Nigeria

Plant Disease ◽  
2019 ◽  
Vol 103 (6) ◽  
pp. 1302-1308 ◽  
Author(s):  
Nomatter Chingandu ◽  
Lelia Dongo ◽  
Osman A. Gutierrez ◽  
Judith K. Brown
Keyword(s):  
Phylogenetic Analyses ◽  
Pcr Amplification ◽  
West African ◽  
Open Reading Frames ◽  
Nucleotide Identity ◽  
Genome Sequences ◽  
Foliar Symptoms ◽  
Field Samples ◽  
Polymerase Chain ◽  
Reading Frames

Cacao swollen shoot disease (CSSD) of Theobroma cacao was reported in Nigeria in 1944; however, no badnaviral genome sequences have been found associated with the symptomatic trees. In 2017, leaf samples (n = 18) were collected from cacao trees from Osun and Oyo, Nigeria showing foliar symptoms that included red vein-banding and shoot swelling, and variable secondary mosaic, mottling, and fern-like pattern symptoms. Abutting primers designed around previously determined 500-bp intergenic region sequences were used for polymerase chain reaction (PCR) amplification. Of the 18 samples, 9 yielded an approximately 7,000-bp, apparently genome-size product. The nine genomes were sequenced and found to encode four open reading frames, and to share 86 to 99% nucleotide identity. Pairwise analysis of the Nigerian genomes with 21 previously reported CSSD badnaviruses, at the complete genome and reverse-transcription ribonuclease H (1,230 bp) sequence levels, indicated 71 to 75 and 72 to 76% nucleotide identity, respectively. Phylogenetic analysis of the nine complete genomes indicated that the closest relatives of the divergent Nigerian isolates were previously described West African CSSD badnaviruses. Based on pairwise comparisons and phylogenetic analyses, the Nigerian CSSD isolates constitute a previously unrecognized Badnavirus sp., herein named Cacao red vein-banding virus (CRVBV). Primers designed based on the CRVBV genome sequences amplified a 1,068-bp fragment from 16 of 18 field samples tested by PCR, suggesting the possible existence of additional CRVBV variants.

scholarly journals Genomic Sequence of Spodoptera frugiperda Ascovirus 1a, an Enveloped, Double-Stranded DNA Insect Virus That Manipulates Apoptosis for Viral Reproduction

2006 ◽  
Vol 80 (23) ◽  
pp. 11791-11805 ◽  
Author(s):  
Dennis K. Bideshi ◽  
Marie-Véronique Demattei ◽  
Florence Rouleux-Bonnin ◽  
Karine Stasiak ◽  
Yeping Tan ◽  
...  
Keyword(s):  
Spodoptera Frugiperda ◽  
Genomic Sequence ◽  
Open Reading Frames ◽  
The Novel ◽  
Fatty Acid Elongase ◽  
Dna Viruses ◽  
Origin And Evolution ◽  
Iridescent Virus ◽  
Double Stranded Dna ◽  
New Hosts

ABSTRACT Ascoviruses (family Ascoviridae) are double-stranded DNA viruses with circular genomes that attack lepidopterans, where they produce large, enveloped virions, 150 by 400 nm, and cause a chronic, fatal disease with a cytopathology resembling that of apoptosis. After infection, host cell DNA is degraded, the nucleus fragments, and the cell then cleaves into large virion-containing vesicles. These vesicles and virions circulate in the hemolymph, where they are acquired by parasitic wasps during oviposition and subsequently transmitted to new hosts. To develop a better understanding of ascovirus biology, we sequenced the genome of the type species Spodoptera frugiperda ascovirus 1a (SfAV-1a). The genome consisted of 156,922 bp, with a G+C ratio of 49.2%, and contained 123 putative open reading frames coding for a variety of enzymes and virion structural proteins, of which tentative functions were assigned to 44. Among the most interesting enzymes, due to their potential role in apoptosis and viral vesicle formation, were a caspase, a cathepsin B, several kinases, E3 ubiquitin ligases, and especially several enzymes involved in lipid metabolism, including a fatty acid elongase, a sphingomyelinase, a phosphate acyltransferase, and a patatin-like phospholipase. Comparison of SfAV-1a proteins with those of other viruses showed that 10% were orthologs of Chilo iridescent virus proteins, the highest correspondence with any virus, providing further evidence that ascoviruses evolved from a lepidopteran iridovirus. The SfAV-1a genome sequence will facilitate the determination of how ascoviruses manipulate apoptosis to generate the novel virion-containing vesicles characteristic of these viruses and enable study of their origin and evolution.

scholarly journals Expression of the Peanut stunt virus Coat Protein Gene Is Essential and Sufficient for Production of Host-Dependent Ribbon-Like Inclusions in Infected Plants

2004 ◽  
Vol 94 (7) ◽  
pp. 722-729 ◽  
Author(s):  
N. S. Bashir ◽  
M. Sanger ◽  
U. Järlfors ◽  
S. A. Ghabrial
Keyword(s):  
Coat Protein ◽  
Leaf Tissue ◽  
Potato Virus X ◽  
Open Reading Frames ◽  
Subgroup Ii ◽  
Enzymelinked Immunosorbent Assay ◽  
Polymerase Chain ◽  
Peanut Stunt Virus ◽  
Virus Coat ◽  
Reading Frames

We previously have reported that infection of tobacco protoplasts or leaf tissue with the cucumovirus Peanut stunt virus (PSV) induced the production of unusual cytoplasmic ribbon-like inclusions. The formation of these novel inclusions is strain-specific, because infection of tobacco with subgroup II PSV strains, but not subgroup I strains, induced the production of inclusions. Furthermore, we have demonstrated that induction of the ribbon-like inclusions maps to PSV subgroup II RNA3, which codes for the coat protein (CP) and movement protein (MP). We have now extended these studies using chimeric constructs containing CP and MP open reading frames (ORFs) from PSV strains ER and W that belong to subgroups I and II, respectively. Additionally, recombinant Potato virus X (PVX) vectors containing translatable and untranslatable PSV CP ORF were constructed. Plants inoculated with infectious chimeric PSV or recombinant PVX transcripts were analyzed for CP expression by enzymelinked immunosorbent assay and reverse transcription-polymerase chain reaction and for inclusion production by electron microscopy. The results of these experiments indicated that translation of the CP ORF alone is essential and sufficient for inclusion production. In immunogold labeling experiments using an antiserum to PSV virions, abundant gold labeling of the inclusions was observed, suggesting that PSV CP is probably a major component of the inclusions. Because inclusion production is host specific, a host factor is likely to be involved. In addition to their diagnostic importance, these novel inclusions may also prove valuable in identifying the host factors that interact with PSV CP.

Detection of Enterotoxigenic Clostridium perfringens in Raw Beef by Polymerase Chain Reaction

1995 ◽  
Vol 58 (2) ◽  
pp. 154-159 ◽  
Author(s):  
LUIS A. BAEZ ◽  
VIJAY K. JUNEJA
Keyword(s):  
Polymerase Chain Reaction ◽  
Clostridium Perfringens ◽  
Direct Detection ◽  
Food Poisoning ◽  
Pcr Amplification ◽  
Oligonucleotide Primer ◽  
Enterotoxin Gene ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Labeled Probe

A polymerase chain reaction (PCR) procedure was developed for direct detection of Clostridium perfringens strains with potential for food poisoning in raw beef samples. An oligonucleotide primer pair was used to amplify a 364 base pair sequence internal to the C. perfringens enterotoxin gene. One milliliter portions of the meat homogenates were inoculated into cooked meat medium (CMM) or reduced Fluid Thioglycollate (FTG) medium and incubated at 37°C. Portions sampled at 2, 4, 6, 8 and 24 h of enrichment were assayed for detection of the enterotoxin sequence by PCR. Amplification of the 364 bp sequence could be detected in 6 h by agarose gel electrophoresis and as early as 2 h by hybridization to a 150 bp digoxigenin (DIG)-labeled probe. To increase the sensitivity of the detection assay a commercial chromosomal deoxyribonucleic acid (DNA) extraction assay was compared with a nested PCR approach. Both methods allowed detection of less than 1 log10 colony forming units (CFU)/g of C. perfringens strains harboring the enterotoxin gene, with no interference with the background microflora present in the raw ground beef.

scholarly journals Insertion and Characterization of the cry1Ia1 Gene in the Potato Cultivar Spunta for Resistance to Potato Tuber Moth

2010 ◽  
Vol 135 (4) ◽  
pp. 317-324 ◽  
Author(s):  
Kelly A. Zarka ◽  
Ria Greyling ◽  
Inge Gazendam ◽  
Dean Olefse ◽  
Kimberly Felcher ◽  
...  
Keyword(s):  
South Africa ◽  
Potato Tuber ◽  
Pcr Amplification ◽  
Potato Cultivar ◽  
Leaf Tissue ◽  
Cauliflower Mosaic Virus ◽  
Open Reading Frames ◽  
Potato Tuber Moth ◽  
Fresh Weight ◽  
Plasmid Genes

Potato tuber moth (Phthorimaea operculella) is a serious pest of potatoes in tropical and subtropical regions of the world, including South Africa. The cry1Ia1 gene (from Bacillus thuringiensis) under the control of the 35S cauliflower mosaic virus promoter was transformed into the potato (Solanum tuberosum) cultivar Spunta to develop a cultivar with resistance to potato tuber moth for release in South Africa. Two transformation events, ‘SpuntaG2’ and ‘SpuntaG3’, were selected and subjected to extensive molecular analyses as required by the regulatory agencies of South Africa. Southern hybridization experiments indicated that ‘SpuntaG2’ and ‘SpuntaG3’ had one and three copies of the cry1Ia1 gene, respectively, and that the gene insertion was stable through multiple clonal generations. Furthermore, the sequence of the cry1Ia1 gene in ‘SpuntaG2’ was compared with the known sequence of the cry1Ia1 gene and found to be identical. Polymerase chain reaction (PCR) amplification using primers for plasmid “backbone” genes demonstrated that ‘SpuntaG2’ contained no backbone plasmid genes, whereas ‘SpuntaG3’ contained several backbone plasmid genes. Therefore, further analyses were limited to ‘SpuntaG2’, and event-specific primers were developed for this cultivar. Analysis of the left and right border regions in ‘SpuntaG2’ demonstrated that the insertion of the cry1Ia1 gene did not disrupt any functional genes nor did it create new open reading frames that encoded proteins with a significant match to the non-redundant sequence database queried by the BLASTP program. Enzyme-linked immunoabsorbent assays (ELISA) tests indicate that the cry1Ia1 gene was expressed at a mean concentration of 2.24 μg·g−1 fresh weight in leaf tissue and 0.12 μg·g−1 fresh weight in tubers. This study demonstrates the extensive molecular characterization that is necessary to apply for deregulation of a genetically modified crop and these data have been used in a regulatory package for the general release of ‘SpuntaG2’.

scholarly journals Characterization of the Genome of the Dairy Lactobacillus helveticus Bacteriophage ΦAQ113

2013 ◽  
Vol 79 (15) ◽  
pp. 4712-4718 ◽  
Author(s):  
Miriam Zago ◽  
Erika Scaltriti ◽  
Lia Rossetti ◽  
Alessandro Guffanti ◽  
Angelarita Armiento ◽  
...  
Keyword(s):  
Genomic Sequence ◽  
Consensus Sequence ◽  
Genome Structure ◽  
Lactobacillus Helveticus ◽  
Open Reading Frames ◽  
Content Type ◽  
Double Stranded Dna ◽  
A Genome ◽  
Health Promoting ◽  
Contractile Tail

ABSTRACTThe complete genomic sequence of the dairyLactobacillus helveticusbacteriophage ΦAQ113 was determined. Phage ΦAQ113 is aMyoviridaebacteriophage with an isometric capsid and a contractile tail. The final assembled consensus sequence revealed a linear, circularly permuted, double-stranded DNA genome with a size of 36,566 bp and a G+C content of 37%. Fifty-six open reading frames (ORFs) were predicted, and a putative function was assigned to approximately 90% of them. The ΦAQ113 genome shows functionally related genes clustered together in a genome structure composed of modules for DNA replication/regulation, DNA packaging, head and tail morphogenesis, cell lysis, and lysogeny. The identification of genes involved in the establishment of lysogeny indicates that it may have originated as a temperate phage, even if it was isolated from natural cheese whey starters as a virulent phage, because it is able to propagate in a sensitive host strain. Additionally, we discovered that the ΦAQ113 phage genome is closely related toLactobacillus gasseriphage KC5a andLactobacillus johnsoniiphage Lj771 genomes. The phylogenetic similarities betweenL. helveticusphage ΦAQ113 and two phages that belong to gut species confirm a possible common ancestral origin and support the increasing consideration ofL. helveticusas a health-promoting organism.

Characterization of a cDNA for Rhesus Monkey Protein C Inhibitor – Evidence for N-Terminal Involvement in Heparin Stimulation

1995 ◽  
Vol 74 (04) ◽  
pp. 1079-1087 ◽  
Author(s):  
Klaus-P Radtke ◽  
José A Fernández ◽  
Bruno O Villoutreix ◽  
Judith S Greengard ◽  
John H Griffin
Keyword(s):  
Rhesus Monkey ◽  
Protein C ◽  
Activated Protein C ◽  
Pcr Amplification ◽  
Amino Acid Sequences ◽  
Heparin Binding ◽  
Reactive Center ◽  
Protein C Inhibitor ◽  
Polymerase Chain

SummarycDNAs for protein C inhibitor (PCI) were cloned from human and rhesus monkey 1 liver RNAs by reverse transcription and polymerase chain reaction (PCR) amplification. Sequencing showed that rhesus monkey and human PCI cDNAs were 93% identical. Predicted amino acid sequences differed at 26 of 387 residues. Pour of these differences (T352M, N359S, R362K, L3631) were in the reactive center loop that is important for inhibitory specificity, and two were in the N-terminal helix (M8T, E13K) that is implicated in glycosaminoglycan binding. PCI in human or rhesus monkey plasma showed comparable inhibitory activity towards human activated protein C in the presence of 10 U/ml heparin. However, maximal acceleration of the inhibition of activated protein C required 5-fold lower heparin concentration for rhesus monkey than for human plasma, consistent with the interpretation that the additional positive charge (E13K) in a putative-heparin binding region increased the affinity for heparin.

scholarly journals DMSO Improves the Ski-Slope Effect in Direct PCR

2021 ◽  
Vol 11 (4) ◽  
pp. 1943
Author(s):  
Joo-Young Kim ◽  
Ju Yeon Jung ◽  
Da-Hye Kim ◽  
Seohyun Moon ◽  
Won-Hae Lee ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Pcr Amplification ◽  
Analytical Techniques ◽  
Direct Pcr ◽  
Dna Profile ◽  
Novel Technologies ◽  
Specific Amplification ◽  
Polymerase Chain ◽  
Dna Profiles

Analytical techniques such as DNA profiling are widely used in various fields, including forensic science, and novel technologies such as direct polymerase chain reaction (PCR) amplification are continuously being developed in order to acquire DNA profiles efficiently. However, non-specific amplification may occur depending on the quality of the crime scene evidence and amplification methods employed. In particular, the ski-slope effect observed in direct PCR amplification has led to inaccurate interpretations of the DNA profile results. In this study, we aimed to reduce the ski-slope effect by using dimethyl sulfoxide (DMSO) in direct PCR. We confirmed that DMSO (3.75%, v/v) increased the amplification yield of large-sized DNA sequences more than that of small-sized ones. Using 50 Korean buccal samples, we further demonstrated that DMSO reduced the ski-slope effect in direct PCR. These results suggest that the experimental method developed in this study is suitable for direct PCR and may help to successfully obtain DNA profiles from various types of evidence at crime scenes.

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