scholarly journals Primer Based Approach for PCR Amplification of High GC Content Gene: Mycobacterium Gene as a Model

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Arbind Kumar ◽  
Jagdeep Kaur
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Codon Optimization ◽  
Gc Content ◽  
Pcr Amplification ◽  
Pcr Conditions

The genome of Mycobacterium is rich in GC content and poses problem in amplification of some genes, especially those rich in the GC content in terminal regions, by standard/routine PCR procedures. Attempts have been made to amplify three GC rich genes of Mycobacterium sp. (Rv0519c and Rv0774c from M. tuberculosis and ML0314c from M. leprae). Out of these three genes, Rv0774c gene was amplified with normal primers under standard PCR conditions, while no amplification was observed in case of Rv0519c and ML0314c genes. In the present investigation a modified primer based approach was successfully used for amplification of GC rich sequence of Rv0519c through codon optimization without changing the native amino acid sequence. The strategy was successfully confirmed by redesigning the standard primers with similar modifications followed by amplification of ML0314c gene.

scholarly journals Identification of cytokine-induced neutrophil chemoattractants (CINC), rat GRO/CINC-2α and CINC-2 β, produced by granulation tissue in culture: purification, complete amino acid sequences and characterization

1994 ◽  
Vol 301 (2) ◽  
pp. 545-550 ◽  
Author(s):  
H Nakagawa ◽  
N Komorita ◽  
F Shibata ◽  
A Ikesue ◽  
K Konishi ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Granulation Tissue ◽  
Pcr Amplification ◽  
Neutrophil Infiltration ◽  
Amino Acid Sequences ◽  
Terminal Amino Acid ◽  
Reverse Transcription Pcr ◽  
Terminal Amino ◽  
The Difference

Four basic neutrophil chemotactic factors (chemokines) have been purified from conditioned medium of granulation tissue obtained from carrageenin-induced inflammation in the rat. On the basis of their N-terminal amino acid sequences, one of the chemokines was identical with rat GRO/cytokine-induced neutrophil chemoattractant (CINC) which we reported previously, and another was identical with rat macrophage inflammatory protein-2 (MIP-2). Two other chemokines were novel chemoattractants related to MIP-2. The novel chemokines are referred to as rat GRO/CINC-2 alpha and CINC-2 beta, and consequently CINC and rat MIP-2 are renamed rat GRO/CINC-1 and CINC-3 respectively. The complete amino acid sequences of purified CINC-2 alpha and CINC-3 were determined by analysis of the fragments isolated from proteinase V8-treated CINCs. The cDNA for CINC-2 beta was cloned by reverse transcription/PCR amplification using specific primers starting with total RNA extracted from lipopolysaccharide-stimulated rat macrophages. A comparison of the amino acid sequence encoded by the cDNA with the N-terminal amino acid sequence of purified CINC-2 beta revealed that mature CINC-2 beta is a 68-residue chemoattractant produced by cleavage of a 32-residue signal peptide. The difference in amino acid sequences between CINC-2 alpha and CINC-2 beta consisted of only three C-terminal residues. Rat GRO/CINC-2 alpha is a major chemokine, and the four purified chemokines have similar chemotactic activity, suggesting that they contribute to neutrophil infiltration into inflammatory sites in rats.

scholarly journals A novel negevirus isolated from Aedes vexans mosquitoes in Finland

2020 ◽  
Vol 165 (12) ◽  
pp. 2989-2992
Author(s):  
Maija T. Suvanto ◽  
Phuoc Truong Nguyen ◽  
Ruut Uusitalo ◽  
Essi M. Korhonen ◽  
Giulia Faolotto ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Boreal Forests ◽  
Sequence Similarity ◽  
Protein A ◽  
Gc Content ◽  
Amino Acid Sequence Similarity ◽  
Aedes Vexans ◽  
Sequence Identity ◽  
Wide Range

Abstract Negeviruses are insect-specific enveloped RNA viruses that have been detected in mosquitoes and sandflies from various geographical locations. Here, we describe a new negevirus from Northern Europe, isolated from pool of Aedes vexans mosquitoes collected in Finland, designated as Mekrijärvi negevirus (MEJNV). MEJNV had a typical negevirus genome organization, is 9,740 nucleotides in length, and has a GC content of 47.53%. The MEJNV genome contains three ORFs, each containing the following identified conserved domains: ORF1 (7,068 nt) encodes a viral methyltransferase, an FtsJ-like methyltransferase, a viral RNA helicase, and an RNA-dependent RNA polymerase, ORF2 (1,242 nt) encodes a putative virion glycoprotein, and ORF3 (660 nt) encodes a putative virion membrane protein. A distinctive feature relative to other currently known negeviruses is a 7-nucleotide-long overlap between ORF1 and ORF2. MEJNV shares the highest sequence identity with Ying Kou virus from China, with 67.71% nucleotide and 75.19% and 59.00% amino acid sequence identity in ORF 1 and ORF 2, respectively. ORF3 had the highest amino acid sequence similarity to Daeseongdong virus 1 and negevirus Nona 1, both with 77.61% identity, and to Ying Kou virus, with 71.22% identity. MEJNV is currently the northernmost negevirus described. Our report supports the view that negeviruses are a globally distributed, diverse group of viruses that can be found from mosquitoes in a wide range of terrestrial biomes from tropical to boreal forests.

Amplification of putative chlorocatechol dioxygenase gene fragments from α- and β-Proteobacteria

1998 ◽  
Vol 44 (5) ◽  
pp. 482-486
Author(s):  
Michelle Leander ◽  
Tatiana Vallaeys ◽  
Roberta Fulthorpe
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Pseudomonas Putida ◽  
Ralstonia Eutropha ◽  
Pcr Amplification ◽  
Dioxygenase Gene ◽  
Dioxygenase Genes

Redundant primers were designed for the PCR amplification of DNA from chlorocatechol dioxygenase genes. These primers were used successfully to amplify 270- to 279-bp fragments from a variety of 2,4-dichlorophenoxyacetate- and chlorobenzoate-degrading strains, including species of Sphingomonas. Three groups of closely related sequences were amplified: one from chlorobenzoate degraders that was 86% similar to the amino acid sequence of the protein coded by the tfdC gene of Ralstonia eutropha JMP134 (pJP4), a second from Sphingomonas strains that was 70% similar to this amino acid sequence, and a third from diverse 2,4-D degraders that showed only 53% similarity to the product coded by tfdC from pJP4 but 88-100% similarity to the product of the tfdC gene of the plasmid pEST4011 from a Pseudomonas putida strain. The primers should be useful in further study of this gene and in tracking a variety of degraders of chloroaromatic compounds in natural systems.Key words: 2,4-dichlorophenoxyacetate, chlorobenzoate, biodegradation, ortho cleavage, detection.

scholarly journals PCR Amplification of Persimmon Fruit β-Galactosidase

HortScience ◽  
1995 ◽  
Vol 30 (4) ◽  
pp. 818D-818
Author(s):  
I.K. Kang ◽  
D.A. Starrett ◽  
S.G. Suh ◽  
J.K. Byun ◽  
K.C. Gross
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Pcr Amplification ◽  
Diospyros Kaki ◽  
Base Pairs ◽  
Persimmon Fruit ◽  
Degenerate Oligonucleotide ◽  
Terminal Amino ◽  
Rapid Amplification ◽  
Antisense Construct

We are studying β-galactosidase (EC 3.2.1.23) in softening persimmon fruit (Diospyros kaki L.f. cv Fuyu) and hope to decrease the rate of softening by inserting an antisense construct of the β-galactosidase gene. The N-terminal amino acid sequence of persimmon fruit β-galactosidase was recently reported. Here we report the cloning of a putative β-galactosidase gene from persimmons. Degenerate oligonucleotide primers were synthesized based on the amino acid sequence. 5′-RACE (rapid amplification of cDNA ends) was done using persimmon Poly A+ mRNA extracted using a phenol: chloroform/LiCl method. Purification was done on an oligo dT-cellulose column. A fragment of roughly 150 base pairs was purified by agarose gel electrophoresis and subcloned into the pCR-Script cloning vector from Stratagene. After sequencing and verifying the insert's identity, it will be isolated and used to screen a persimmon fruit cDNA library currently being constructed. Ultimately this cDNA clone will be used to make an antisense β-galactosidase construct that will be transformed into persimmon.

scholarly journals Passaging impact of H9N2 avian influenza virus in hamsters on its pathogenicity and genetic variability

2014 ◽  
Vol 8 (05) ◽  
pp. 570-580 ◽  
Author(s):  
Houssam A. Shaib ◽  
Nelly Cochet ◽  
Thierry Ribeiro ◽  
Afif M Abdel Nour ◽  
Georges Nemer ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Avian Influenza ◽  
Mammalian Cells ◽  
Pcr Amplification ◽  
Amino Acid Sequences ◽  
Influenza Viruses ◽  
H9n2 Virus ◽  
World Health ◽  
The Impact

Introduction: Avian influenza viruses of the H9N2 subtype have been reported to cause human infections. This study demonstrates the impact of nasal viral passaging of avian H9N2 in hamsters on its cross species-pathogenic adaptability and variability of amino acid sequences of the hemagglutinin (HA) and neuraminidase (NA) stalk. Methodology: Three intranasal passagings of avian H9N2 in hamsters P1, P2, and P3 were accomplished. Morbidity signs and lesions were observed three days post viral inoculation. The HA test was used for presumptive detection of H9N2 virus in the trachea and lungs of the hamsters challenged with the differently passaged viruses. Different primers were used for PCR amplification of the HA1 and NA stalk regions of the differently passaged H9N2 viruses, followed by sequence alignment. Results: The morbidity signs indicated low pathogenicity of the differently passaged H9N2 viruses in hamsters. The frequency of gross and microscopic lesions in the tracheas and lungs were insignificantly different among hamsters challenged with the differently passaged H9N2 viruses (p > 0.05). There was 100% similarity in the amino acid sequence of the HA gene of most passaged viruses. The amino acid sequence of the neuraminidase in the third passaged H9N2 virus recovered from lungs showed a R46P mutation that might have a role in the pathogenic adaptability of P3 viruses in hamsters’ lungs. Conclusions: The apparent adaptation of avian H9N2 virus to mammalian cells is in agreement with the World Health Organization’s alertness for a possible public health threat by this adaptable virus.

cDNA sequence and tissue-specific expression of an anionic flax peroxidase

Genome ◽  
1994 ◽  
Vol 37 (1) ◽  
pp. 137-147 ◽  
Author(s):  
Franz Omann ◽  
Normand Beaulieu ◽  
Hugh Tyson
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Pcr Amplification ◽  
Glycosylation Site ◽  
Northern Analysis ◽  
Sequence Comparisons ◽  
Specific Expression ◽  
Amino Acid Region ◽  
Linum Usitatissimum L ◽  
Plant Peroxidases

A flax (Linum usitatissimum L.) λgt10 cDNA library was screened with a probe coding for the amino terminus of a flax peroxidase. The probe was generated by PCR amplification of the library with one of the λgt10 sequencing primers and a mixed oligonucleotide derived from a well-conserved amino acid region (distal heme ligand) found in all plant peroxidases. A positive clone (FLXPER2) was isolated and characterized. The cDNA contains 1153 nucleotides, excluding the poly(A) tail, and encodes a mature protein of 297 amino acids with a molecular mass of 31.9 kDa. The mature protein's amino acid sequence contains three highly conserved regions, two of which contain histidine ligands for the heme group. The deduced amino acid sequence has nine cysteine residues. Eight are identically located to those of horseradish C peroxidase, which participate in four disulfide bridges; these cysteines are highly conserved in all plant peroxidases. One potential N-glycosylation site (Asn-X-Thr/Ser) is present. The predicted pI value of 4.5 identifies FLXPER2 as an anionic peroxidase. Northern blot analysis shows that its mRNA expression is unique to stem tissue. Amino acid sequence comparisons show high similarity between FLXPER2 and peanut, rice, and tobacco peroxidases.Key words: flax peroxidase cDNA, PCR, Northern analysis, sequence relationships.

The staining pattern of the tropomyosin sequence is displayed in a new paracrystal

1986 ◽  
Vol 44 ◽  
pp. 150-151
Author(s):  
M.K. Lamvik ◽  
L.L. Klatt
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Staining Pattern ◽  
Banding Patterns ◽  
Mass Thickness ◽  
New Form

Tropomyosin paracrystals have been used extensively as test specimens and magnification standards due to their clear periodic banding patterns. The paracrystal type discovered by Ohtsuki1 has been of particular interest as a test of unstained specimens because of alternating bands that differ by 50% in mass thickness. While producing specimens of this type, we came across a new paracrystal form. Since this new form displays aligned tropomyosin molecules without the overlaps that are characteristic of the Ohtsuki-type paracrystal, it presents a staining pattern that corresponds to the amino acid sequence of the molecule.

Isolation and Structural Charaderization of a Potent Inhibitor of Coagulation Factor Xa from the Leech Haementeria ghilianii

1989 ◽  
Vol 61 (03) ◽  
pp. 437-441 ◽  
Author(s):  
Cindra Condra ◽  
Elka Nutt ◽  
Christopher J Petroski ◽  
Ellen Simpson ◽  
P A Friedman ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Salivary Gland ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Western Blot ◽  
Potent Inhibitor ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Sds Page ◽  
Bovine Factor

SummaryThe present work reports the discovery and charactenzation of an anticoagulant protein in the salivary gland of the giant bloodsucking leech, H. ghilianii, which is a specific and potent inhibitor of coagulation factor Xa. The inhibitor, purified to homogeneity, displayed subnanomolar inhibition of bovine factor Xa and had a molecular weight of approximately 15,000 as deduced by denaturing SDS-PAGE. The amino acid sequence of the first 43 residues of the H. ghilianii derived inhibitor displayed a striking homology to antistasin, the recently described subnanomolar inhibitor of factor Xa isolated from the Mexican leech, H. officinalis. Antisera prepared to antistasin cross-reacted with the H. ghilianii protein in Western Blot analysis. These data indicate that the giant Amazonian leech, H. ghilianii, and the smaller Mexican leech, H. officinalrs, have similar proteins which disrupt the normal hemostatic clotting mechanisms in their mammalian host’s blood.

Paris I Dysfibrinogenemia: A Point Mutation in Intron 8 Results in Insertion of a 15 Amino Acid Sequence in the Fibrinogen γ-Chain

1993 ◽  
Vol 69 (03) ◽  
pp. 217-220 ◽  
Author(s):  
Jonathan B Rosenberg ◽  
Peter J Newman ◽  
Michael W Mosesson ◽  
Marie-Claude Guillin ◽  
David L Amrani
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Point Mutation ◽  
Genomic Dna ◽  
Comparative Sequence Analysis ◽  
Chain Gene ◽  
Molecular Defect ◽  
Nucleotide Position ◽  
Normal Individuals ◽  
Polymerase Chain

SummaryParis I dysfibrinogenemia results in the production of a fibrinogen molecule containing a functionally abnormal γ-chain. We determined the basis of the molecular defect using polymerase chain reaction (PCR) to amplify the γ-chain region of the Paris I subject’s genomic DNA. Comparative sequence analysis of cloned PCR segments of normal and Paris I genomic DNA revealed only an A→G point mutation occurring at nucleotide position 6588 within intron 8 of the Paris I γ-chain gene. We examined six normal individuals and found only normal sequence in this region, indicating that this change is not likely to represent a normal polymorphism. This nucleotide change leads to a 45 bp fragment being inserted between exons 8 and 9 in the mature γparis I chain mRNA, and encodes a 15 amino acid insert after γ350 [M-C-G-E-A-L-P-M-L-K-D-P-C-Y]. Alternative splicing of this region from intron 8 into the mature Paris I γ-chain mRNA also results after translation into a substitution of S for G at position γ351. Biochemical studies of 14C-iodoacetamide incorporation into disulfide-reduced Paris I and normal fibrinogen corroborated the molecular biologic predictions that two additional cysteine residues exist within the γpariS I chain. We conclude that the insertion of this amino acid sequence leads to a conformationallyaltered, and dysfunctional γ-chain in Paris I fibrinogen.

Sign in / Sign up

Export Citation Format

Share Document