scholarly journals Triticum mosaic virus: A New Virus Isolated from Wheat in Kansas

Plant Disease ◽  
2008 ◽  
Vol 92 (5) ◽  
pp. 808-817 ◽  
Author(s):  
Dallas L. Seifers ◽  
T. J. Martin ◽  
Tom L. Harvey ◽  
John P. Fellers ◽  
James P. Stack ◽  
...  
Keyword(s):  
Coat Protein ◽  
Mosaic Virus ◽  
Polyclonal Antibodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cesium Chloride ◽  
High Plains ◽  
Flight Mass Spectrometry ◽  
Sds Page ◽  
Systemic Symptoms

In 2006, a mechanically-transmissible and previously uncharacterized virus was isolated in Kansas from wheat plants with mosaic symptoms. The physiochemical properties of the virus were examined by purification on cesium chloride density gradients, electron microscopy, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), sequencing of the nucleotides and amino acids of the coat protein, and immunological reactivity. Purified preparations contained flexuous, rod-shaped particles that resembled potyviruses. The coat protein was estimated from SDS-PAGE to have a mass of approximately 35 kDa. Its amino acid sequence, as deduced from DNA sequencing of cloned, reverse-transcribed viral RNA and separately determined by time-of-flight mass spectrometry, was most closely related (49% similarity) to Sugarcane streak mosaic virus, a member of the Tritimovirus genus of the family Potyviridae. The virus gave strong positive reactions during enzyme-linked immunosorbent assays using polyclonal antibodies raised against purified preparations of the cognate virus but gave consistent negative reactions against antibodies to Wheat streak mosaic virus (WSMV), other wheat potyviruses, and the High Plains virus. When the virus was inoculated on the WSMV-resistant wheat cv. RonL, systemic symptoms appeared and plant growth was diminished significantly in contrast with WSMV-inoculated RonL. Taken together, the data support consideration of this virus as a new potyvirus, and the name Triticum mosaic virus (TriMV) is proposed.

scholarly journals Association of a Virus with Wheat Displaying Yellow Head Disease Symptoms in the Great Plains

Plant Disease ◽  
2005 ◽  
Vol 89 (8) ◽  
pp. 888-895 ◽  
Author(s):  
Dallas L. Seifers ◽  
Tom L. Harvey ◽  
T. J. Martin ◽  
Steve Haber ◽  
Y.-M. She ◽  
...  
Keyword(s):  
Great Plains ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Mass Spectrometry Analysis ◽  
High Plains ◽  
Spectrometry Analysis ◽  
Yellow Head Virus ◽  
Flight Mass Spectrometry ◽  
Sds Page ◽  
Leaf Symptoms

Wheat with yellow head disease (YHD) (yellow heads and mosaic leaf symptoms) has been observed in Kansas since 1997. A pathogen was transmitted from the infected wheat to maize by vascular puncture inoculation and to Nicotiana benthamiana by rub inoculation. The original infected wheat and infected maize and N. benthamiana test plants all produced a unique 32- to 34-kDa protein when analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Time-of-flight mass spectrometry analysis of the unique 32- to 34-kDa protein showed that the amino acid sequence was most closely related to the nucleoprotein of Rice hoja blanca virus, indicating that the virus causing YHD symptoms in wheat is a tenuivirus. Antiserum made to this protein failed to react with extracts made from healthy wheat or wheat infected with Wheat streak mosaic virus or the High Plains virus. The antiserum did react to extracts made from symptomatic wheat, maize, and N. benthamiana, shown by SDS-PAGE to contain the unique protein, and to extracts of wheat with YHD symptoms from Kansas, North Dakota, South Dakota, and Oklahoma. The name Wheat yellow head virus is proposed for this virus.

scholarly journals Identification of Variants of the High Plains virus Infecting Wheat in Kansas

Plant Disease ◽  
2009 ◽  
Vol 93 (12) ◽  
pp. 1265-1274 ◽  
Author(s):  
Dallas L. Seifers ◽  
T. J. Martin ◽  
Tom L. Harvey ◽  
S. Haber ◽  
O. Krokhin ◽  
...  
Keyword(s):  
Amino Acid ◽  
Coat Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Amino Acid Sequences ◽  
Enzyme Linked Immunosorbent Assay ◽  
High Plains ◽  
Protein Amino Acid ◽  
Sds Page ◽  
Virus Isolates

The properties of two virus isolates (U04-82 and U04-83) obtained from two wheat (Triticum aestivum) plants expressing mosaic symptoms were investigated using enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), time-of-flight mass spectrometry (TOFMS), and infection of wheat with resistance to Wheat streak mosaic virus (WSMV). The coat protein mass was estimated by SDS-PAGE as approximately 32 kDa for U04-82 and 30 kDa for U04-83. The amino acid sequence of the coat protein of U04-82 was 99.6 and 85.5% identical to two isolates, ABC58222 and TX96, respectively, of High Plains virus (HPV) described from Texas. U04-82 was transmitted by wheat curl mites and caused significant yield reductions in wheat resistant to WSMV. U04-83 was actually two distinct virus isolates whose capsid protein amino acid sequences were only 57 and 50% similar to that of TX96. Antiserum prepared to a synthetic peptide from the sequence of the U04-83 isolate recognized the two U04-83 isolates, but not the U04-82 isolate.

scholarly journals Characterization of a Novel Potyvirus Isolated from Maize in Israel

2000 ◽  
Vol 90 (5) ◽  
pp. 505-513 ◽  
Author(s):  
D. L. Seifers ◽  
R. Salomon ◽  
V. Marie-Jeanne ◽  
B. Alliot ◽  
P. Signoret ◽  
...  
Keyword(s):  
Amino Acid ◽  
Mosaic Virus ◽  
Pennisetum Glaucum ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Amino Acid Sequences ◽  
Maize Dwarf Mosaic Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Flight Mass Spectrometry ◽  
Sds Page

A potyvirus (proposed name of Zea mosaic virus [ZeMV]) isolated from maize in Israel was analyzed by serology, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of capsid proteins, symptomatology, and sequencing. Parts of the nuclear inclusion b, coat protein, and 3′ regions were sequenced; the amino acid sequence of ZeMV capsid was determined by time-of-flight mass spectrometry (TOFMS). The results of these analyses were compared with those of similar analyses of the following potyviruses: Maize dwarf mosaic virus (MDMV), Sugarcane mosaic virus strain MDB (SCMV-MDB), Johnsongrass mosaic virus(JGMV), Sorghum mosaic virus (SrMV), and an isolate of MDMV from Israel. Indirect enzyme-linked immunosorbent assay using ZeMV antiserum detected only ZeMV, and reciprocal tests using MDMV, JGMV, or SrMV antisera failed to detect ZeMV. ZeMV cross-reacted weakly when SCMV-MDB antiserum was used. The mass of ZeMV capsid was determined to be 36,810 Da by SDS-PAGE and 34,216 Da by TOFMS. The ZeMV systemically infected johnsongrass (Sorghum halepense), but did not infect oat (Avena sativa), pearl millet (Pennisetum glaucum), barley (Hordeum vulgare), or rye (Secale cereale). Necrosis was caused in 19 sorghum lines by SrMV, in 15 by ZeMV, in 14 by MDMV, and in 5 by JGMV and SCMV-MDB. The nucleic acid and amino acid sequences of ZeMV clearly showed that it is not a strain of JGMV, MDMV, SCMV, or SrMV.

scholarly journals First Record of Barley yellow striate mosaic virus Affecting Wheat Summer-Nurseries in Syria

Plant Disease ◽  
2004 ◽  
Vol 88 (1) ◽  
pp. 83-83 ◽  
Author(s):  
Khaled M. Makkouk ◽  
Safaa G. Kumari ◽  
Widad Ghulam ◽  
Nouran Attar
Keyword(s):  
Mosaic Virus ◽  
Barley Yellow Dwarf Virus ◽  
Polyclonal Antibodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
First Record ◽  
Structural Protein ◽  
Western Blots ◽  
N Protein ◽  
Yellow Dwarf Virus

A limited survey to identify virus diseases affecting wheat in summer nurseries in agricultural stations in southern Syria was conducted during October 2002. A total of 94 bread and durum wheat samples with symptoms suggestive of virus infection (stripping, stunting, and yellowing) were collected. All samples were tested for the presence of four viruses by tissue-blot immunoassay (2) at the Virology Laboratory of ICARDA, Aleppo, Syria using the following polyclonal antibodies: Barley stripe mosaic virus (BSMV); Barley yellow dwarf virus-PAV (BYDV-PAV) and Wheat streak mosaic virus (WSMV) from the Virology Laboratory at ICARDA; and Barley yellow striate mosaic virus (BYSMV) isolated from Italy (BYSMV-Italy) and provided by M. Conti, Instituto di Fitovirologia applicata, Turino, Italy. Serological results obtained indicated that BYSMV was the most commonly encountered virus (78.7%) followed by BYDV-PAV (22.3%), whereas, BSMV and WSMV were not detected in any of the samples tested. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by western blots, purified BYSMV preparations were observed to contain a 47-kDa structural protein typical of the N protein of Rhabdoviruses that reacted strongly with three BYSMV antisera (BYSMV-Italy, BYSMV-Lebanon [4], and BYSMV-Morocco [1]). Samples that reacted with BYSMV antisera were transmitted from wheat to wheat, barley, and oat plants by the planthopper Laodelphax striatella (Fallen) (Hemiptera: family Delphacidae) in a persistent manner, and the major symptoms of BYSMV on cereal crops were stripping and stunting. BYDV-PAV has been reported from Syria earlier (3) but to our knowledge, this is the first report of BYSMV affecting wheat in Syria. References: (1) B. E. Lockhart et al. Plant Dis. 70:1113, 1986. (2) K. M. Makkouk and A. Comeau. Eur. J. Plant Pathol. 100:71, 1994. (3) K. M. Makkouk et al. Phytopathol. Mediterr. 28:164, 1989. (4) K. M. Makkouk et al. Plant Dis. 85:446, 2001.

scholarly journals A New Method for Extraction and Analysis of Ricin Samples through MALDI-TOF-MS/MS

Toxins ◽  
2019 ◽  
Vol 11 (4) ◽  
pp. 201 ◽  
Author(s):  
Roberto B. Sousa ◽  
Keila S. C. Lima ◽  
Caleb G. M. Santos ◽  
Tanos C. C. França ◽  
Eugenie Nepovimova ◽  
...  
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Amino Acid Sequences ◽  
Intact Protein ◽  
Toxic Activity ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Flight Mass Spectrometry ◽  
Sds Page ◽  
Tof Ms

We report for the first time the efficient use of accelerated solvent extraction (ASE) for extraction of ricin to analytical purposes, followed by the combined use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and MALDI-TOF MS/MS method. That has provided a fast and unambiguous method of ricin identification for in real cases of forensic investigation of suspected samples. Additionally, MALDI-TOF MS was applied to characterize the presence and the toxic activity of ricin in irradiated samples. Samples containing ricin were subjected to ASE, irradiated with different dosages of gamma radiation, and analyzed by MALDI-TOF MS/MS for verification of the intact protein signal. For identification purposes, samples were previously subjected to SDS-PAGE, for purification and separation of the chains, followed by digestion with trypsin, and analysis by MALDI-TOF MS/MS. The results were confirmed by verification of the amino acid sequences of some selected peptides by MALDI-TOF MS/MS. The samples residual toxic activity was evaluated through incubation with a DNA substrate, to simulate the attack by ricin, followed by MALDI-TOF MS/MS analyses.

Comparative HPLC, SDS-PAGE, and Immunoblot Analyses of Dental Enamel Proteins

1996 ◽  
Vol 10 (2) ◽  
pp. 150-158 ◽  
Author(s):  
O.H. Ryu ◽  
C.-C. Hu ◽  
J.P. Simmer
Keyword(s):  
Rna Splicing ◽  
Polyclonal Antibodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sequence Divergence ◽  
Dental Enamel ◽  
Reversed Phase ◽  
Enamel Matrix ◽  
Sds Page ◽  
Exon 4

The primary structures of amelogenins expressed from different genes vary because of DNA sequence divergence and variations in alternative RNA splicing. The pattern of splicing is unique for each amelogenin gene yet investigated, even when two copies of the gene are expressed in the same cell. Despite the high conservation of amelogenin sequences, diversity in the pattern of RNA splicing leads to significant differences in the number and character of amelogenin isoforms in the developing enamel matrix. Since conservation of molecular structure is an indicator of functional significance, we compared enamel protein preparations from rat, porcine, rabbit, and opossum developing tooth organs. Enamel extracts were fractionated by reversed-phase high-performance liquid chromatography (HPLC) and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Western blot analyses were performed with polyclonal antibodies raised against recombinant murine amelogenin and the polypeptide encoded by murine exon 4. The opossum enamel extract produced the simplest chromatogram, suggesting that fewer proteins are secreted into the developing enamel matrix. The predominant opossum amelogenin has an apparent molecular mass of 28 kDa and reacts strongly with the recombinant amelogenin antibody but is not recognized by the murine exon 4 antibody. Opossum amelogenin mRNA was amplified with murine amelogenin primers specific for the amino- and carboxyl-terminal coding regions. The mobility of the amplification products on 4% agarose gels indicates that the leucine-rich amelogenin polypeptide (LRAP) is expressed in the opossum and that the major amelogenin is larger than its homologue in the mouse. We conclude that the alternative splicing of amelogenins pre-dates the metatherian and eutherian divergence over 100 million years ago.

scholarly journals KARAKTER MOLEKULER CHRYSANTHEMUM B CARLAVIRUS (CVB) ISOLAT KRISAN (DENDRANTHEMA GRANDIFLORA KITAM) DI INDONESIA

2011 ◽  
Vol 8 (2) ◽  
pp. 138-145
Author(s):  
I G. R. M. Temaja ◽  
G. Suastika ◽  
S.H. Hidayat ◽  
U. Kartosuwondo
Keyword(s):  
Coat Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Analysis ◽  
Protein Band ◽  
Dendranthema Grandiflora ◽  
Research Activities ◽  
Sds Page ◽  
Close Relationship ◽  
Molecular Characters

Molecular characterstics of Chrysanthemum B Carlavirus (CVB) isolated from chrysanthemum (Dendranthema grandiflora Kitam) in Indonesia. Chrysanthemum B Carlavirus (CVB) belongs to Carlavirus genus which type species is Carnation latent virus (CLV). Since CVB is considered a new plant virus in chrysanthemum plantation in Indonesia, a study on its molecular characters is required. The objectives of the study are: 1) to determine molecular characters of CVB; 2) to study genetic diversity among CVB isolates collected from different geographic regions in Indonesia. The research activities cover virus purifications, electron microscope observation, coat protein analysis by SDS PAGE, and nucleic acid analysis. The result of virus purification demonstrated a high purity level with ratio value of A260/A280 =1.22. The total pure virus produced from 200 g of fresh material is 6.250 mg. Purified virus preparation yielded rather straight rod and flexuous virus particles of about 685 nm long and 12 nm wide. Coat protein analysis with sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed specific protein band of approximately 34 kDa. Specific DNA fragment of 739 bp was successfully amplified from chrysanthemum infected by CVB Cianjur, Medan, Malang and Bali isolates. CVB isolated from Cianjur, Medan, Malang and Bali have similarity 85-99%. Based on analysis using PAUP 4.10 program, Cianjur, Medan, Malang and Bali isolates belong to the same group with CVB isolates originated from India (Chattisgarh and Jammu isolates). Cianjur isolate has close relationship to Medan isolate, however Bali isolate showed a close relationship with Malang isolate.

scholarly journals Expression and purification of recombinant coat protein of sugarcane mosaic virus from Indonesian isolate as an antigen for antibody production

2019 ◽  
Vol 24 (1) ◽  
pp. 57
Author(s):  
Natalia Tri Astuti ◽  
Nurmalasari Darsono ◽  
Suvia Widyaningrum ◽  
Widhi Dyah Sawitri ◽  
Sri Puji Astuti ◽  
...  
Keyword(s):  
Coat Protein ◽  
Mosaic Virus ◽  
Polyclonal Antibodies ◽  
Molecular Study ◽  
Insoluble Fraction ◽  
Sugarcane Mosaic Virus ◽  
Gene Encoding ◽  
Iptg Induction ◽  
Sds Page ◽  
Saccharum Sp

Sugarcane mosaic virus (SCMV, genus Potyvirus, family Potyviridae) is a prominent pathogen of sugarcane (Saccharum sp. hybrids). It can cause losses in susceptible varieties, in crop as well as sugar production, economically. Although it has been studied in major sugar-producing countries, research on the definement of SCMV from Indonesian isolates based on molecular study has been very limited. This study aimed to obtain a proper recombinant antigens emanating from coat protein of SCMV from Indonesian isolate in order to produce polyclonal antibodies that cann be used for immunodiagnosis assays in a subsequent study. A gene-encoding coat protein of SCMV (CP-SCMV) was amplified using RT-PCR and cloned into vector pJET1.2. The cDNA was inserted into 6X His-tag expression plasmid of pET28a(+) and over-expressed in Escherichia coli BL21(DE3) to produce a recombinant protein. The highest expression was found in 0.1M IPTG induction media for 5 h at 37oC. SDS-PAGE analysis clarified that the recombinant CP-SCMV remained as an insoluble fraction. Purifications was carried out by the affinity Ni-NTA resin, followed by electroelution to obtain a highly purified protein. To meet the quality requirements of a proper antigen, the highly purified protein was concentrated. A molecular weight of the rCP-SCMV (approximately 40 kDa) was clearly observed by 10% SDS-PAGE at the concentration of 16.184 mg/mL. 

scholarly journals Enhancement of Binding Affinity of Anti-Hapten Polyclonal IgG Recognizing Mitragynine using Affinity Purification

2021 ◽  
Vol 29 (4) ◽  
Author(s):  
Radhiahtul Raehan Mustafa ◽  
Rashidah Sukor ◽  
Siti Mariam Mohd Nor ◽  
Nazamid Saari ◽  
Farina Mustaffa Kamal ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Biomedical Applications ◽  
Polyclonal Antibodies ◽  
Affinity Purification ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sds Page ◽  
Elution Peak ◽  
Polyclonal Igg ◽  
Aromatic Ether

Antibodies are glycoproteins found in peritoneal fluid, serum, and blood. The antibody-based assay has been used for broad applications such as immunodiagnostic and other biomedical applications. Depending on the intended application, a highly purified polyclonal antibody could be used as an alternative. Purification of antibodies from anti-sera has been proven as one of the methods to enhance the binding affinity of antibodies towards its antigen. We report herein the enhancement of the binding affinity of anti-hapten polyclonal IgG recognizing mitragynine using affinity purification. Serum from the terminal bleed of New Zealand White (NZW) rabbits immunized with mitragynine conjugated with cationized– bovine serum albumin at methyl ester (C22-MG-cBSA), or aromatic ether modification (C9-MG-cBSA) were subjected to HiTrap Protein G affinity purification using fast protein liquid chromatography (FPLC). The elution peak from chromatography fractions was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Here, we report the binding of polyclonal antibodies produced from inoculation of either C22-MG-cBSA or C9-MG-cBSA immunogens of which mitragynine-ovalbumin (MG-OVA) was used as coating antigen in the ELISA assay. Non purified anti-sera from C22-MG-cBSA-inoculated rabbits showed higher titer than C9-MG-cBSA at 1/128 000 and 1/32 000 dilutions, respectively. The affinity of purified poly-IgGs from rabbits immunized with C22-MG-cBSA showed a mean Kd value of 7.965 × 10-6 μM, which was lower than those immunized with C9-MG-cBSA at mean Kd of 1.390 × 10-4 μM. In addition, the purified poly- IgGs showed higher binding towards MG-OVA than non-purified anti-sera at comparable protein concentrations. These results indicated that the higher binding affinity of purified polyclonal IgG is due to the reduced competition among polyclonal antibodies with non- IgG proteins that co-existed in the non-purified anti-sera after the affinity purification.

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