scholarly journals A Point Mutation within the Replicase Gene Differentially Affects Coronavirus Genome versus Minigenome Replication

2005 ◽  
Vol 79 (24) ◽  
pp. 15016-15026 ◽  
Author(s):  
Carmen Galán ◽  
Luis Enjuanes ◽  
Fernando Almazán
Keyword(s):  
Amino Acid ◽  
Point Mutation ◽  
Cdna Clone ◽  
Reverse Genetics ◽  
Point Mutations ◽  
Full Length ◽  
Transmissible Gastroenteritis Virus ◽  
Replicase Gene ◽  
Full Length Cdna

ABSTRACT During the construction of the transmissible gastroenteritis virus (TGEV) full-length cDNA clone, a point mutation at position 637 that was present in the defective minigenome DI-C was maintained as a genetic marker. Sequence analysis of the recovered viruses showed a reversion at this position to the original virus sequence. The effect of point mutations at nucleotide 637 was analyzed by reverse genetics using a TGEV full-length cDNA clone and cDNAs from TGEV-derived minigenomes. The replacement of nucleotide 637 of TGEV genome by a T, as in the DI-C sequence, or an A severely affected virus recovery from the cDNA, yielding mutant viruses with low titers and small plaques compared to those of the wild type. In contrast, T or A at position 637 was required for minigenome rescue in trans by the helper virus. No relationship between these observations and RNA secondary-structure predictions was found, indicating that mutations at nucleotide 637 most likely had an effect at the protein level. Nucleotide 637 occupies the second codon position at amino acid 108 of the pp1a polyprotein. This position is predicted to map in the N-terminal polyprotein papain-like proteinase (PLP-1) cleavage site at the p9/p87 junction. Replacement of G-637 by A, which causes a drastic amino acid change (Gly to Asp) at position 108, affected PLP-1-mediated cleavage in vitro. A correlation was found between predicted cleaving and noncleaving mutations and efficient virus rescue from cDNA and minigenome amplification, respectively.

scholarly journals Beta-centractin: characterization and distribution of a new member of the centractin family of actin-related proteins.

1994 ◽  
Vol 5 (12) ◽  
pp. 1301-1310 ◽  
Author(s):  
S W Clark ◽  
O Staub ◽  
I B Clark ◽  
E L Holzbaur ◽  
B M Paschal ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cdna Clone ◽  
Expressed Sequence Tag ◽  
Full Length ◽  
Northern Analysis ◽  
Related Protein ◽  
Two Dimensional Gel Electrophoresis ◽  
Full Length Cdna ◽  
Dynactin Complex ◽  
Expressed Sequence

An examination of human-expressed sequence tags indicated the existence of an isoform of centractin, an actin-related protein localized to microtubule-associated structures. Using one of these tags, we isolated and determined the nucleotide sequence of a full-length cDNA clone. The protein encoded represents the first example of multiple isoforms of an actin-related protein in a single organism. Northern analysis using centractin-specific probes revealed three species of mRNA in HeLa cells that could encode centractin isoforms. One mRNA encodes the previously-identified centractin (now referred to as alpha-centractin). The full-length cDNA clone isolated using the expressed sequence tag encodes a new member of the centractin family, beta-centractin. A probe specific for alpha-centractin hybridized to the third species of mRNA observed (referred to as gamma-centractin). Comparisons of Northern blots of human tissues indicated that alpha-centractin and beta-centractin mRNAs are equally distributed in all populations of mRNA examined, whereas the expression of gamma-centractin appears to be tissue specific. The amino acid sequence of beta-centractin, deduced from the cDNA, indicates a 91% identity with alpha-centractin, increasing to 96% similarity when conservative amino acid changes are taken into account. As antibodies previously raised against alpha-centractin reacted only poorly with beta-centractin, new antibodies were produced and combined with two-dimensional gel electrophoresis to discriminate the two isoforms. Using this system, the subcellular distribution of the alpha- and beta-isoforms were determined. Both isoforms were found predominantly in the cytosolic fraction as a part of a previously identified 20S complex (referred to as the dynactin complex) with no evidence for a free pool of either isoform. The isoforms were found in a constant ratio of approximately 15:1 (alpha:beta) in the dynactin complex.

scholarly journals Infectious genomic RNA of Rhopalosiphum padi virus transcribed in vitro from a full-length cDNA clone

Virology ◽  
2008 ◽  
Vol 375 (2) ◽  
pp. 401-411 ◽  
Author(s):  
Sandhya Boyapalle ◽  
Randy J. Beckett ◽  
Narinder Pal ◽  
W. Allen Miller ◽  
Bryony C. Bonning
Keyword(s):  
Cdna Clone ◽  
Rhopalosiphum Padi ◽  
Full Length ◽  
Genomic Rna ◽  
Full Length Cdna

scholarly journals Structure of a full-length cDNA clone for the preproα2(I) chain of human type I procollagen. Comparison with the chicken gene confirms unusual patterns of gene conservation

1988 ◽  
Vol 252 (3) ◽  
pp. 633-640 ◽  
Author(s):  
H Kuivaniemi ◽  
G Tromp ◽  
M L Chu ◽  
D J Prockop
Keyword(s):  
Amino Acid ◽  
Cdna Clone ◽  
Full Length ◽  
Total Amino Acid ◽  
Type I ◽  
Amino Acid Residues ◽  
Full Length Cdna ◽  
Human Type ◽  
Type I Procollagen ◽  
I Gene

A cDNA clone from a human placental library was found to consist of an essentially full-length cDNA of 4.6 kb for the prepro alpha 2(I) chain of type I procollagen. Nucleotide sequencing of the 5′-end of the cDNA provided a sequence of 1617 nucleotide residues and codons for 539 amino acid residues not previously defined. Comparison of the complete structure of the prepro alpha 2(I) cDNA with previously reported sequences for the chicken pro alpha 2(I) gene indicated that 83% of 1366 total amino acid residues were conserved. In the alpha-chain domain 84% of 1014 amino acid residues were conserved. Also, there was conservation of the previously noted preference for U and C in the third position of codons for glycine, proline and alanine. One major difference between the human and the chicken prepro alpha 2(I) chain was that the human chain contained 21 fewer proline residues, an observation that probably explains why the triple helix of human type I procollagen unfolds at temperatures that are 1-2 degrees C lower. In parallel experiments, sequencing of intron-exon boundaries for nine exons of genomic subclones confirmed and extended previous observations that the pro alpha 2(I) gene, like other genes from fibrillar collagens, has an unusual 54-base pattern of exon sizes that is highly conserved through evolution.

scholarly journals Construction of a full-length infectious cDNA clone of swine vesicular disease virus strain NET/1/92 and analysis of new antigenic variants derived from it

2000 ◽  
Vol 81 (11) ◽  
pp. 2763-2769 ◽  
Author(s):  
J. M. J. Rebel ◽  
C. H. Leendertse ◽  
A. Dekker ◽  
F. van Poelwijk ◽  
R. J. M. Moormann
Keyword(s):  
Amino Acid ◽  
Disease Virus ◽  
Cdna Clone ◽  
Parent Strain ◽  
Biological Properties ◽  
Infectious Cdna Clone ◽  
Full Length ◽  
Swine Vesicular Disease Virus ◽  
Vesicular Disease

The Dutch swine vesicular disease virus (SVDV) isolate NET/1/92 was one of the first isolates belonging to a new SVDV antigenic group. This strain was completely sequenced and was shown to have 93% similarity with the UKG/27/72 isolate. To enable antigenicity, replication, maturation and pathogenicity studies of NET/1/92, an infectious full-length cDNA clone, designated pSVD146, was prepared. The in vitro and in vivo biological properties of the virus derived from pSVD146 were studied by analysing antigenicity, plaque morphology, growth curves and virulence in pigs. The epitopes of newly prepared monoclonal antibodies were roughly mapped by fusion-PCR. Fine mapping of epitopes at the amino acid level was achieved by introducing single amino acid mutations in pSVD146. Two new amino acids important in epitope formation were located in VP1; one was mapped in the C-terminal end and the second is thought to be located in the H–I loop. Growth curve and plaque sizes in vitro were similar between virus derived from pSVD146 and the parent wild-type virus. In virulence studies in pigs, the lesions score, neutralization titres and the seroconversion rates were comparable between virus derived from pSVD146 and the parent strain. Since virus derived from pSVD146 had the same biological properties as the parent strain NET/1/92, the full-length infectious cDNA clone pSVD146 will be very useful in studies of the antigenicity, virulence, pathogenesis, maturation and replication of SVDV.

scholarly journals The complete amino acid sequence of a pea (Pisum sativum) seed lipoxygenase predicted from a near full-length cDNA

1988 ◽  
Vol 253 (3) ◽  
pp. 915-918 ◽  
Author(s):  
P M Ealing ◽  
R Casey
Keyword(s):  
Amino Acid ◽  
Pisum Sativum ◽  
Amino Acid Sequence ◽  
Amino Acid Identity ◽  
Full Length ◽  
Predicted Amino Acid Sequence ◽  
Protein Coding ◽  
Full Length Cdna ◽  
Coding Regions

A near full-length cDNA for a pea (Pisum sativum) seed lipoxygenase was isolated and sequenced. It has a protein coding sequence (2583 bp), 5′ (59 bp) and 3′ (191 bp) non-coding regions, and a poly(A) tail (20 bp). The predicted amino acid sequence indicates a polypeptide of Mr 97,628 that shows about 86% amino acid identity with a soya-bean lipoxygenase 3 protein sequence [Yenofsky, Fine & Liu (1988) Mol. Gen. Genet. 211, 215-222]. The cDNA directs the transcription of mRNA that can be translated to give an anti-lipoxygenase-precipitable polypeptide in vitro.

scholarly journals Generation of an Infectious Clone of VR-2332, a Highly Virulent North American-Type Isolate of Porcine Reproductive and Respiratory Syndrome Virus

2003 ◽  
Vol 77 (6) ◽  
pp. 3702-3711 ◽  
Author(s):  
H. S. Nielsen ◽  
G. Liu ◽  
J. Nielsen ◽  
M. B. Oleksiewicz ◽  
A. Bøtner ◽  
...  
Keyword(s):  
Growth Kinetics ◽  
Cdna Clone ◽  
North American ◽  
Molecular Mechanisms ◽  
Full Length ◽  
Respiratory Syndrome Virus ◽  
Full Length Cdna ◽  
Kinetics Of ◽  
Highly Virulent

ABSTRACT A full-length cDNA clone of the prototypical North American porcine reproductive and respiratory syndrome virus (PRRSV) isolate VR-2332 was assembled in the plasmid vector pOK12. To rescue infectious virus, capped RNA was transcribed in vitro from the pOK12 clone and transfected into BHK-21C cells. The supernatant from transfected monolayers were serially passaged on Marc-145 cells and porcine pulmonary alveolar macrophages. Infectious PRRSV was recovered on Marc-145 cells as well as porcine pulmonary macrophages; thus, the cloned virus exhibited the same cell tropism as the parental VR-2332 strain. However, the cloned virus was clearly distinguishable from the parental VR-2332 strain by an engineered marker, a BstZ17I restriction site. The full-length cDNA clone had 11 nucleotide changes, 2 of which affected coding, compared to the parental VR-2332 strain. Additionally, the transcribed RNA had an extra G at the 5′ end. To examine whether these changes influenced viral replication, we examined the growth kinetics of the cloned virus in vitro. In Marc-145 cells, the growth kinetics of the cloned virus reflected those of the parental isolate, even though the titers of the cloned virus were consistently slightly lower. In experimentally infected 5.5-week-old pigs, the cloned virus produced blue discoloration of the ears, a classical clinical symptom of PRRSV. Also, the seroconversion kinetics of pigs infected with the cloned virus and VR-2332 were very similar. Hence, virus derived from the full-length cDNA clone appeared to recapitulate the biological properties of the highly virulent parental VR-2332 strain. This is the first report of an infectious cDNA clone based on American-type PRRSV. The availability of this cDNA clone will allow examination of the molecular mechanisms behind PRRSV virulence and attenuation, which might in turn allow the production of second-generation, genetically engineered PRRSV vaccines.

Recombinant expression and characterization of lemon (Citrus limon) peroxidase

2020 ◽  
Vol 27 ◽  
Author(s):  
Veda P. Pandey ◽  
Apoorvi Tyagi ◽  
Shagoofa Ali ◽  
Kusum Yadav ◽  
Anurag Yadav ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Plant Defense ◽  
Cdna Clone ◽  
Protein Function ◽  
In Silico ◽  
Full Length ◽  
Function Annotation ◽  
Full Length Cdna ◽  
Protein Function Annotation

Background: Class III plant peroxidases play important role in a number of physiological processes in plant such as lignin biosynthesis, suberization, cell wall biosynthesis, reactive oxygen species metabolism and plant defense against pathogens. Peroxidases are also of significance in several industrial applications. In view of this, the production and identification of novel peroxidases having resistance towards temperature, pH, salts is desirable. Objective: The objective of the present work was to clone and characterize a novel plant peroxidase suitable for industrial application. Methods: A full length cDNA clone of lemon peroxidase was isolated using PCR and RACE approaches, characterized and heterologously expressed in Escherichia coli using standard protocols. The expressed peroxidase was purified using Ni-NTA agarose column and biochemically characterized using standard protocols. The peroxidase was also in-silico characterized at nucleotide as well as protein levels using standard protocols. Results: A full length cDNA clone of lemon peroxidase was isolated and expressed heterologously expressed in Escherichia coli. The expressed recombinant lemon peroxidase (LPRX) was activated by in-vitro refolding and purified. The purified LPRX exhibited pH and temperature optima of pH 7.0 and 50°C, respectively. The LPRX was found to be activated by metal ions (Na+ , Ca2+, Mg2+ and Mn2+) at lower concentration. The expressional analysis of the transcripts suggested involvement of lemon peroxidase in plant defense. The lemon peroxidase was in silico modelled and docked with the substrates guaiacol, and pyrogallol and results show the favourability of pyrogallol over guaiacol, which is in agreement with the in-vitro findings. The protein function annotation analyses suggested the involvement of lemon peroxidase in the phenylpropanoid biosynthesis pathway and plant defense mechanisms. Conclusion: Based on the biochemical characterization, the purified peroxidase was found to be resistant towards the salts and thus, might be a good candidate for industrial exploitation. The in-silico protein function annotation and transcript analyses highlighted the possible involvement of the lemon peroxidase in plant defense response.

Genome structure of Sagiyama virus and its relatedness to other alphaviruses

Microbiology ◽  
2000 ◽  
Vol 81 (5) ◽  
pp. 1353-1360 ◽  
Author(s):  
Yukio Shirako ◽  
Yuka Yamaguchi
Keyword(s):  
Amino Acid ◽  
Nucleotide Sequence ◽  
Cdna Clone ◽  
Consensus Sequence ◽  
Genome Structure ◽  
Nucleotide Sequences ◽  
Full Length ◽  
Nonstructural Proteins ◽  
Full Length Cdna ◽  
Original Stock

Sagiyama virus (SAG) is a member of the genus Alphavirus in the family Togaviridae, isolated in Japan from mosquitoes in 1956. We determined the complete nucleotide sequence of the SAG genomic RNA from the original stock virus which formed a mixture of plaques with different sizes, and that from a full-length cDNA clone, pSAG2, infectious RNA transcripts from which formed uniform large plaques on BHK-21 cells. The SAG genome was 11698 nt in length exclusive of the 3′ poly(A) tail. Between the complete nucleotide sequences of the full-length cDNA clone, pSAG2, and the consensus sequence from the original stock virus, there were nine amino acid differences; two each in nsP1, nsP2 and E1, and three in E2, some of which may be responsible for plaque phenotypic variants in the original virus stock. SAG was most closely related to Ross River virus among other alphaviruses fully sequenced, with amino acid sequence identities of 86% in the nonstructural proteins and of 83% in the structural proteins. The 3′ terminal 280 nt region of SAG was 82% identical to that of Barmah Forest virus, which was otherwise not closely related to SAG. Comparison of the nucleotide sequence of SAG with partial nucleotide sequences of Getah virus (GET), which was originally isolated in Malaysia in 1955 and is closely related to SAG in serology and in biology, showed near identity between the two viruses, suggesting that SAG is a strain of GET.

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