scholarly journals Characterization and Construction of Functional cDNA Clones of Pariacoto Virus, the First Alphanodavirus Isolated outside Australasia

2000 ◽  
Vol 74 (11) ◽  
pp. 5123-5132 ◽  
Author(s):  
Karyn N. Johnson ◽  
Jean-Louis Zeddam ◽  
L. Andrew Ball
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Capsid Protein ◽  
Cultured Cells ◽  
Geographic Origin ◽  
Amino Acid Sequences ◽  
Cdna Clones ◽  
Homologous Proteins ◽  
Genomic Rnas ◽  
Rna Genome

ABSTRACT Pariacoto virus (PaV) was recently isolated in Peru from the Southern armyworm (Spodoptera eridania). PaV particles are isometric, nonenveloped, and about 30 nm in diameter. The virus has a bipartite RNA genome and a single major capsid protein with a molecular mass of 39.0 kDa, features that support its classification as aNodavirus. As such, PaV is the firstAlphanodavirus to have been isolated from outside Australasia. Here we report that PaV replicates in wax moth larvae and that PaV genomic RNAs replicate when transfected into cultured baby hamster kidney cells. The complete nucleotide sequences of both segments of the bipartite RNA genome were determined. The larger genome segment, RNA1, is 3,011 nucleotides long and contains a 973-amino-acid open reading frame (ORF) encoding protein A, the viral contribution to the RNA replicase. During replication, a 414-nucleotide long subgenomic RNA (RNA3) is synthesized which is coterminal with the 3′ end of RNA1. RNA3 contains a small ORF which could encode a protein of 90 amino acids similar to the B2 protein of other alphanodaviruses. RNA2 contains 1,311 nucleotides and encodes the 401 amino acids of the capsid protein precursor α. The amino acid sequences of the PaV capsid protein and the replicase subunit share 41 and 26% identity with homologous proteins of Flock house virus, the best characterized of the alphanodaviruses. These and other sequence comparisons indicate that PaV is evolutionarily the most distant of the alphanodaviruses described to date, consistent with its novel geographic origin. Although the PaV capsid precursor is cleaved into the two mature capsid proteins β and γ, the amino acid sequence at the cleavage site, which is Asn/Ala in all other alphanodaviruses, is Asn/Ser in PaV. To facilitate the investigation of PaV replication in cultured cells, we constructed plasmids that transcribed full-length PaV RNAs with authentic 5′ and 3′ termini. Transcription of these plasmids in cells recreated the replication of PaV RNA1 and RNA2, synthesis of subgenomic RNA3, and translation of viral proteins A and α.

scholarly journals A spatial similarity of stereochemical environments formed by amino acid residues defines a common epitope of two non-homologous proteins

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Kentaro Nakashima ◽  
Shintaro Iwashita ◽  
Takehiro Suzuki ◽  
Chieko Kato ◽  
Toshiyuki Kohno ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Cultured Cells ◽  
Amino Acid Sequences ◽  
Cross Reaction ◽  
Amino Acid Residues ◽  
Homologous Proteins ◽  
Spatial Similarity ◽  
Target Molecules ◽  
The Cross

Abstract It is critical for development of high-quality antibodies in research and diagnostics to predict accurately their cross-reactivities with “off-target” molecules, which potentially induce false results. Herein, we report a good example of such a cross-reactivity for an off-target due to a stereochemical environment of epitopes, which does not simply depend on amino acid sequences. We found that significant subpopulation of a polyclonal peptide antibody against Bcnt (Bucentaur) (anti-BCNT-C antibody) cross-reacted with a completely different protein, glutamine synthetase (GS), and identified four amino acids, GYFE, in its C-terminal region as the core amino acids for the cross-reaction. Consistent with this finding, the anti-BCNT-C antibody strongly recognized endogenously and exogenously expressed GS in tissues and cultured cells by Western blotting and immunohistochemistry. Furthermore, we elucidated that the cross-reaction is caused by a spatial similarity between the stereochemical environments formed by amino acid residues, including the GYFE of GS and the GYIE of Bcnt, rather than by their primary sequences. These results suggest it is critical to comprehensively analyze antibody interactions with target molecules including off-targets with special attention to the physicochemical environments of epitope-paratope interfaces to decrease the risk of false interpretations of results using antibodies in science and clinical applications.

scholarly journals Nucleotide and derived amino acid sequences of a cDNA coding for pre-uteroglobin from the lung of the hare (Lepus capensis)

1986 ◽  
Vol 235 (3) ◽  
pp. 895-898 ◽  
Author(s):  
M S López de Haro ◽  
A Nieto
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Nucleotide Sequence ◽  
Amino Acid Sequence ◽  
Amino Acid Sequences ◽  
Untranslated Regions ◽  
Coding Region ◽  
Homologous Proteins ◽  
Lepus Capensis ◽  
Rabbit Gene

An almost full-length cDNA coding for pre-uteroglobin from hare lung was cloned and sequenced. The derived amino acid sequence indicated that hare pre-uteroglobin contained 91 amino acids, including a signal peptide of 21 residues. Comparison of the nucleotide sequence of hare pre-uteroglobin cDNA with that previously reported for the rabbit gene indicated five silent point substitutions and six others leading to amino acid changes in the coding region. The untranslated regions of both pre-uteroglobin mRNAs were very similar. The amino acid changes observed are discussed in relation to the different progesterone-binding abilities of both homologous proteins.

scholarly journals Comparison of Ambisense M RNA of Watermelon Silver Mottle Virus with Other Tospoviruses

1998 ◽  
Vol 88 (4) ◽  
pp. 351-358 ◽  
Author(s):  
Fang-Hua Chu ◽  
Shyi-Dong Yeh
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Consensus Sequence ◽  
Chenopodium Quinoa ◽  
Distinct Species ◽  
Mottle Virus ◽  
Impatiens Necrotic Spot Virus ◽  
Cdna Clones ◽  
Genomic Rnas ◽  
Watermelon Silver Mottle Virus

Double-stranded genomic RNAs (dsRNAs) extracted from Chenopodium quinoa infected with watermelon silver mottle virus (WSMV) were similar to those of tomato spotted wilt virus (TSWV, serogroup I) and impatiens necrotic spot virus (INSV, serogroup III), except that the S dsRNA of WSMV is 0.75 and 0.6 kbp longer than those of TSWV and INSV, respectively. The complete nucleotide sequence of the genomic M RNA of WSMV was determined from cDNA clones generated from separated M dsRNA. The M RNA is 4,880 nucleotides in length with two open reading frames (ORFs) in an ambisense organization. The M RNA-encoded nonstructural (NSm) ORF located on the viral strand encodes a protein of 312 amino acids (35 kDa), and the G1/G2 ORF located on the viral complementary strand encodes a protein of 1,121 amino acids (127.6 kDa). The RNA probe corresponding to the NSm or G1/G2 ORF of WSMV failed to hybridize with the M dsRNAs of TSWV and INSV. Comparison of M and S RNAs of WSMV, TSWV, INSV, and peanut bud necrosis virus (PBNV, serogroup IV) revealed a consensus sequence of eight nucleotides of 5′-AGAGCAAU…-3′ at their 5′ ends and 5′-…AUUGCUCU-3′ at their 3′ ends. The low overall nucleotide identities (56.4 to 56.9%) of the M RNA and the low amino acid identities of the NSm and G1/G2 proteins (30.5 to 40.9%) with those of TSWV and INSV indicate that WSMV belongs to the Tospovirus genus but is phylogenetically distinct from viruses in serogroups I and III. The M RNA of WSMV shares a nucleotide identity of 79.6% with that of PBNV, and the two viruses share 83.4 and 88.7% amino acid identities for their NSm and G1/G2 proteins, respectively. It is concluded that they are two related but distinct species of serogroup IV. In addition to the viral or viral complementary full-length M RNA, two putative RNA messages for the NSm gene and the G1/G2 gene, 1.0 and 3.4 kb, respectively, were detected from the total RNA extracted from WSMV-infected tissue of Nicotiana benthamiana. The 1.0- and 3.4-kb RNAs were also detected in the viral RNAs extracted from purified nucleocapsids, suggesting that the putative messages of the M RNA of WSMV can also be encapsidated by the nucleocapsid protein.

scholarly journals 088 Differential Expression of Expansin Genes Isolated from Sweet Cherry (Prunus avium L.) during Fruit Ripening

HortScience ◽  
2000 ◽  
Vol 35 (3) ◽  
pp. 403F-404
Author(s):  
Zhencai Wu ◽  
Paul A. Wiersma
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Fruit Development ◽  
Sweet Cherry ◽  
Prunus Avium ◽  
Expression Patterns ◽  
Amino Acid Sequences ◽  
Cdna Clones ◽  
Low Level ◽  
Expansin Gene

Expansins are a class of proteins that stimulate the extension of plant cell walls. Expansins have been found in nearly all growing plant tissues, such as hycopotyls, young seedlings, fibers, internodes, flower petals, and ripening fruits. We isolated two full-length expansin cDNA clones, Pruav-Exp1 and Pruav-Exp2, from sweet cherry (Prunus avium L.) fruit. Pruav-Exp1 has 1048 nucleotides encoding 254 amino acids, while Pruav-Exp2 has 1339 nucleotides encoding 250 amino acids. Deduced amino acid sequences of sweet cherry Pruav-Exp1 and Pruav-Exp2 share 72% identity. A Blast search of the GenBank database with the deduced amino acid sequences of Pruav-Exp1 and Pruav-Exp2 indicated a high sequence identity with other plant expansin genes. Interestingly, Pruav-Exp1 shares 99% identity of amino acid sequence with that of apricot expansin Pav-Exp1. Fragments from the 3' ends of Pruav-Exp1 and Pruav-Exp2 were cloned to generate gene-specific probes. These probes were used to study expansin gene expression in different tissues and during fruit development. Northern blot analysis showed different mRNA expression patterns for each gene. The mRNA of Pruav-Exp1 was expressed at the pink and ripe stages, but not at the early green and yellow stages of fruit development. The mRNA of Pruav-Exp2 was present earlier, from a low level in yellow expanding fruit, increasing to a high level at the pink stage and remaining at this level through the ripe stage. Both mRNAs were also expressed at a low level in flower, but not present in other tissues such as roots, leaves and peduncles. Our study indicates an expansin gene family is present in sweet cherry and suggests that two expansin genes may have different roles during fruit development and ripening.

scholarly journals A Conserved Capsid Protein Surface Domain of Cucumber Mosaic Virus Is Essential for Efficient Aphid Vector Transmission

2002 ◽  
Vol 76 (19) ◽  
pp. 9756-9762 ◽  
Author(s):  
Sijun Liu ◽  
Xiaohua He ◽  
Gyungsoon Park ◽  
Caroline Josefsson ◽  
Keith L. Perry
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Capsid Protein ◽  
Wild Type Virus ◽  
Cdna Clones ◽  
Wild Type ◽  
Vector Transmission ◽  
Aphid Vector

ABSTRACT A prominent feature on the surfaces of virions of Cucumber mosaic virus (CMV) is a negatively charged loop structure (the βH-βI loop). Six of 8 amino acids in this capsid protein loop are highly conserved among strains of CMV and other cucumoviruses. Five of these amino acids were individually changed to alanine or lysine (an amino acid of opposite charge) to create nine mutants (the D191A, D191K, D192A, D192K, L194A, E195A, E195K, D197A, and D197K mutants). Transcripts of cDNA clones were infectious when they were mechanically inoculated onto tobacco, giving rise to symptoms of a mottle-mosaic typical of the wild-type virus (the D191A, D191K, D192A, E195A, E195K, and D197A mutants), a systemic necrosis (the D192K mutant), or an atypical chlorosis with necrotic flecking (the L194A mutant). The mutants formed virions and accumulated to wild-type levels, but eight of the nine mutants were defective in aphid vector transmission. The aspartate-to-lysine mutation at position 197 interfered with infection; the only recovered progeny (the D197K∗ mutant) harbored a second-site mutation (denoted by the asterisk) of alanine to glutamate at position 193, a proximal site in the βH-βI loop. Since the disruption of charged amino acid residues in the βH-βI loop reduces or eliminates vector transmissibility without grossly affecting infectivity or virion formation, we hypothesize that this sequence or structure has been conserved to facilitate aphid vector transmission.

scholarly journals Combinations of two amino acids (Ala40 and Phe75 or Ser40 and Tyr75) in the coat protein of apple chlorotic leaf spot virus are crucial for infectivity

2007 ◽  
Vol 88 (9) ◽  
pp. 2611-2618 ◽  
Author(s):  
Hajime Yaegashi ◽  
Masamichi Isogai ◽  
Hiroko Tajima ◽  
Teruo Sano ◽  
Nobuyuki Yoshikawa
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Coat Protein ◽  
Leaf Spot ◽  
Amino Acid Sequences ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Cdna Clones ◽  
Spot Virus ◽  
Chlorotic Leaf

Amino acid sequences of apple chlorotic leaf spot virus (ACLSV) coat protein (CP) were compared between 12 isolates from apple, plum and cherry, and 109 cDNA clones that were amplified directly from infected apple tissues. Phylogenetic analysis based on the amino acid sequences of CP showed that the isolates and cDNA clones were separated into two major clusters in which the combinations of the five amino acids at positions 40, 59, 75, 130 and 184 (Ala40-Val59-Phe75-Ser130-Met184 or Ser40-Leu59-Tyr75-Thr130-Leu184) were highly conserved within each cluster. Site-directed mutagenesis using an infectious cDNA clone of ACLSV indicated that the combinations of two amino acids (Ala40 and Phe75 or Ser40 and Tyr75) are necessary for infectivity to Chenopodium quinoa plants by mechanical inoculation. Moreover, an agroinoculation assay indicated that the substitution of a single amino acid (Ala40 to Ser40 or Phe75 to Tyr75) resulted in extreme reduction in the accumulation of viral genomic RNA, double-stranded RNAs and viral proteins (movement protein and CP) in infiltrated tissues, suggesting that the combinations of the two amino acids at positions 40 and 75 are important for effective replication in host plant cells.

scholarly journals Roles of the C-Terminal Amino Acids of Non-Hexameric Helicases: Insights from Escherichia coli UvrD

2021 ◽  
Vol 22 (3) ◽  
pp. 1018
Author(s):  
Hiroaki Yokota
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Single Molecule ◽  
Underlying Mechanism ◽  
Amino Acid Sequences ◽  
E Coli ◽  
X Ray Crystallography ◽  
Terminal Amino

Helicases are nucleic acid-unwinding enzymes that are involved in the maintenance of genome integrity. Several parts of the amino acid sequences of helicases are very similar, and these quite well-conserved amino acid sequences are termed “helicase motifs”. Previous studies by X-ray crystallography and single-molecule measurements have suggested a common underlying mechanism for their function. These studies indicate the role of the helicase motifs in unwinding nucleic acids. In contrast, the sequence and length of the C-terminal amino acids of helicases are highly variable. In this paper, I review past and recent studies that proposed helicase mechanisms and studies that investigated the roles of the C-terminal amino acids on helicase and dimerization activities, primarily on the non-hexermeric Escherichia coli (E. coli) UvrD helicase. Then, I center on my recent study of single-molecule direct visualization of a UvrD mutant lacking the C-terminal 40 amino acids (UvrDΔ40C) used in studies proposing the monomer helicase model. The study demonstrated that multiple UvrDΔ40C molecules jointly participated in DNA unwinding, presumably by forming an oligomer. Thus, the single-molecule observation addressed how the C-terminal amino acids affect the number of helicases bound to DNA, oligomerization, and unwinding activity, which can be applied to other helicases.

scholarly journals Cloning and sequence analysis of cDNAs encoding the α1, α2 and α3 chains of mouse collagen VI

1993 ◽  
Vol 291 (3) ◽  
pp. 787-792 ◽  
Author(s):  
R Z Zhang ◽  
T C Pan ◽  
R Timpl ◽  
M L Chu
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequences ◽  
Untranslated Regions ◽  
Cdna Libraries ◽  
Cdna Clones ◽  
Collagen Vi ◽  
Central Segment ◽  
Glycosylation Sites ◽  
Key Features ◽  
Triple Helical Domain

cDNA clones encoding the alpha 1, alpha 2 and alpha 3 chains of mouse collagen VI have been isolated by screening cDNA libraries with the corresponding human probes. The composite cDNAs for the alpha 1, alpha 2, and alpha 3 chains are 2.5, 1.6 and 2.9 kb in size respectively. The alpha 1 and alpha 2 cDNAs encode the C-terminal portions of the chains as well as the entire 3′-untranslated regions, while the alpha 3 cDNAs encode a central segment of 959 amino acids flanking the triple-helical domain. The deduced amino acid sequences share 86-88% identity with the human counterparts and 67-73% identity with the chicken equivalents. Alignment of the deduced amino acid sequences of mouse, human and chicken collagens reveal that the key features of the protein, including the cysteine residues, imperfections in the Gly-Xaa-Xaa regions, Arg-Gly-Asp sequences and potential N-glycosylation sites, are mostly conserved.

scholarly journals Molecular cloning of two human liver 3 α-hydroxysteroid/dihydrodiol dehydrogenase isoenzymes that are identical with chlordecone reductase and bile-acid binder

1994 ◽  
Vol 299 (2) ◽  
pp. 545-552 ◽  
Author(s):  
Y Deyashiki ◽  
A Ogasawara ◽  
T Nakayama ◽  
M Nakanishi ◽  
Y Miyabe ◽  
...  
Keyword(s):  
Bile Acid ◽  
Amino Acid ◽  
Human Liver ◽  
Polyclonal Antibodies ◽  
Amino Acid Sequences ◽  
Cdna Clones ◽  
Amino Acid Residues ◽  
Human Bile ◽  
Coding Regions ◽  
Computer Based

Human liver contains two dihydrodiol dehydrogenases, DD2 and DD4, associated with 3 alpha-hydroxysteroid dehydrogenase activity. We have raised polyclonal antibodies that cross-reacted with the two enzymes and isolated two 1.2 kb cDNA clones (C9 and C11) for the two enzymes from a human liver cDNA library using the antibodies. The clones of C9 and C11 contained coding sequences corresponding to 306 and 321 amino acid residues respectively, but lacked 5′-coding regions around the initiation codon. Sequence analyses of several peptides obtained by enzymic and chemical cleavages of the two purified enzymes verified that the C9 and C11 clones encoded DD2 and DD4 respectively, and further indicated that the sequence of DD2 had at least additional 16 residues upward from the N-terminal sequence deduced from the cDNA. There was 82% amino acid sequence identity between the two enzymes, indicating that the enzymes are genetic isoenzymes. A computer-based comparison of the cDNAs of the isoenzymes with the DNA sequence database revealed that the nucleotide and amino acid sequences of DD2 and DD4 are virtually identical with those of human bile-acid binder and human chlordecone reductase cDNAs respectively.

scholarly journals Synaptopodin: An Actin-associated Protein in Telencephalic Dendrites and Renal Podocytes

1997 ◽  
Vol 139 (1) ◽  
pp. 193-204 ◽  
Author(s):  
Peter Mundel ◽  
Hans W. Heid ◽  
Thomas M. Mundel ◽  
Meike Krüger ◽  
Jochen Reiser ◽  
...  
Keyword(s):  
Dendritic Spines ◽  
Cultured Cells ◽  
Rat Kidney ◽  
Amino Acid Sequences ◽  
Cdna Clones ◽  
Globular Domain ◽  
Postnatal Maturation ◽  
Human And Mouse

Synaptopodin is an actin-associated protein of differentiated podocytes that also occurs as part of the actin cytoskeleton of postsynaptic densities (PSD) and associated dendritic spines in a subpopulation of exclusively telencephalic synapses. Amino acid sequences determined in purified rat kidney and forebrain synaptopodin and derived from human and mouse brain cDNA clones show no significant homology to any known protein. In particular, synaptopodin does not contain functional domains found in receptor-clustering PSD proteins. The open reading frame of synaptopodin encodes a polypeptide with a calculated Mr of 73.7 kD (human)/74.0 kD (mouse) and an isoelectric point of 9.38 (human)/9.27 (mouse). Synaptopodin contains a high amount of proline (∼20%) equally distributed along the protein, thus virtually excluding the formation of any globular domain. Sequence comparison between human and mouse synaptopodin revealed 84% identity at the protein level. In both brain and kidney, in vivo and in vitro, synaptopodin gene expression is differentiation dependent. During postnatal maturation of rat brain, synaptopodin is first detected by Western blot analysis at day 15 and reaches maximum expression in the adult animal. The exclusive synaptopodin synthesis in the telencephalon has been confirmed by in situ hybridization, where synaptopodin mRNA is only found in perikarya of the olfactory bulb, cerebral cortex, striatum, and hippocampus, i.e., the expression is restricted to areas of high synaptic plasticity. From these results and experiments with cultured cells we conclude that synaptopodin represents a novel kind of proline-rich, actin-associated protein that may play a role in modulating actin-based shape and motility of dendritic spines and podocyte foot processes.

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