scholarly journals Overexpression of the mulberry latex gene MaMLX-Q1 enhances defense against Plutella xylostella in Arabidopsis thaliana

2017 ◽  
Vol 69 (2) ◽  
pp. 269-276 ◽  
Author(s):  
Yan Liu ◽  
Dongfeng Ji ◽  
Jine Chen ◽  
Tianbao Lin ◽  
Jia Wei ◽  
...  
Keyword(s):  
Plutella Xylostella ◽  
Plant Protection ◽  
Degenerate Primers ◽  
Reading Frame ◽  
Binding Domains ◽  
Heterogeneous Expression ◽  
Protein Group ◽  
Rapid Amplification ◽  
Family 19 Chitinase ◽  
Race Pcr

Purified mulberry latex chitinase (MLX) has a role in defense against some lepidopteran insects. In this study, a full length chitinase gene, MaMLX-Q1, of 1405 bp with a 1140 bp open reading frame, was obtained from mulberry leaves by the degenerate primers and rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR) procedure. The gene encoded a mature protein with the predicted molecular mass of 39.38 kDa and an isoelectric point (pI) of 6.43; it contained two chitin-binding domains and a hydrolase family 19 chitinase domain. Sequence alignment and phylogenetic analysis grouped it in the class I chitinase protein group. Heterogeneous expression of this MaMLX-Q1 in Arabidopsis showed non-visible alterations in growth phenotype, except for the higher transcriptional expression of MaMLX-Q1 when compared to that of wild-type Arabidopsis. This ectopic MaMLX-Q1 exhibited toxicity to the growth and development of Plutella xylostella larvae, causing significantly lower weight gain and higher mortality. These results indicate an application of MaMLX-Q1 as an insecticide for plant protection.

A second expressed kininogen gene in mice

2006 ◽  
Vol 26 (2) ◽  
pp. 152-157 ◽  
Author(s):  
Edward G. Shesely ◽  
Chun-Bo Hu ◽  
François Alhenc-Gelas ◽  
Pierre Meneton ◽  
Oscar A. Carretero
Keyword(s):  
Molecular Weight ◽  
Open Reading Frame ◽  
Low Molecular Weight ◽  
High Molecular Weight Kininogen ◽  
Rt Pcr ◽  
Reading Frame ◽  
Rna Ligase ◽  
Mouse Tissues ◽  
Rapid Amplification ◽  
Race Pcr

We isolated PCR, RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE-PCR)-, and RT-PCR-generated clones from mouse kininogen family transcripts. DNA sequencing indicated that the clones were from two distinct genes. One set (K1) is from the previously reported mouse kininogen gene. The second set (K2) has an open reading frame, is 93% identical to K1 in the overlapping nucleotide sequence, and, unlike T-kininogens in the rat, encodes a bradykinin motif identical to K1. We discovered that K2 exists with two different 5′ ends. We used RT-PCR to determine the distribution and relative abundance of K1 and K2 mRNA in mouse tissues. K2 is transcribed and K1 and K2 are generally both expressed in the same tissues; however, they differ in their regulation of the alternative splicing event that yields either low-molecular-weight kininogen (LMWK) or high-molecular-weight kininogen (HMWK). For example, in the liver K1 is expressed as both HMWK and LMWK, whereas K2 is only expressed as LMWK. Conversely, in the kidney K2 is strongly expressed as both HMWK and LMWK, whereas K1 is not expressed as HMWK and expressed only very weakly as LMWK.

Cloning and functional characterization of a monoterpene synthase gene from Eleutherococcus trifoliatus

Holzforschung ◽  
2015 ◽  
Vol 69 (2) ◽  
pp. 163-171 ◽  
Author(s):  
Kuan-Feng Huang ◽  
Yi-Ru Lee ◽  
Yen-Hsueh Tseng ◽  
Sheng-Yang Wang ◽  
Fang-Hua Chu
Keyword(s):  
Functional Characterization ◽  
Open Reading Frame ◽  
Terpene Synthase ◽  
Gas Chromatography Mass Spectrometry ◽  
Degenerate Primers ◽  
Reading Frame ◽  
Monoterpene Synthase ◽  
Young Leaves ◽  
Rapid Amplification

AbstractEleutherococcus trifoliatusalso known as the three-leavedEleutherococcus, a member of the Araliaceae (ginseng) family, is commonly used in traditional Chinese medicine. Recently, many studies have demonstrated the bioactivities of the secondary metabolites inE. trifoliatus. In this study, a monoterpene synthase fromE. trifoliatushas been characterized. A pair of degenerate primers was designed and a fragment with conserved region of terpene synthase (TPS) was obtained. After 5′- and 3′-rapid amplification of cDNA ends (RACE), the full-length cDNA was obtained. The gene designatedEtLIMcontains an open reading frame of 1752 bp with a predicated molecular mass of 67.3 kDa. It was expressed in young leaves, stems, and drupes. The product ofEtLIMhas been identified by gas chromatography/mass spectrometry (GC/MS) as limonene.

Molecular cloning and assessment of the immunocontraceptive potential of the zona pellucida subunit 3 from Brandt's vole (Microtus brandti)

2006 ◽  
Vol 18 (3) ◽  
pp. 331 ◽  
Author(s):  
Hui Li ◽  
Yun-shang Piao ◽  
Zhi-bin Zhang ◽  
Christopher M. Hardy ◽  
Lyn A. Hinds
Keyword(s):  
Amino Acid ◽  
Zona Pellucida ◽  
Keyhole Limpet Hemocyanin ◽  
Amino Acid Residues ◽  
Igg Antibody ◽  
Reading Frame ◽  
Rapid Amplification ◽  
Ovarian Pathology ◽  
Race Pcr ◽  
Brandt’S Vole

A full-length cDNA encoding Brandt’s vole (Microtus brandti) zona pellucida glycoprotein subunit 3 (vZP3) was isolated using rapid amplification of cDNA ends–polymerase chain reaction (RACE-PCR). The cDNA contains an open reading frame of 1254 nucleotides encoding a polypeptide of 418 amino acid residues. The deduced amino acid sequence of vZP3 revealed high overall homology with hamster (82.1%), mouse (81.3%) and rat (80.6%). A synthetic vZP3 peptide corresponding to amino acid residues 328–343 was conjugated to keyhole limpet hemocyanin (KLH-vZP3328–343) and used to immunise female Brandt’s voles in order to test the efficacy of this peptide as a contraceptive antigen. High IgG antibody levels to the vZP3328–343 peptide were present in the sera of female voles immunised with KLH-vZP3328–343 and these also cross-reacted to the zona pellucida in ovaries of Brandt’s vole. The fertility of the KLH-vZP3328–343-immunised voles was reduced by 50% compared with controls without evidence of significant ovarian pathology.

scholarly journals d-Ala-d-Ser VanN-Type Transferable Vancomycin Resistance in Enterococcus faecium

2011 ◽  
Vol 55 (10) ◽  
pp. 4606-4612 ◽  
Author(s):  
François Lebreton ◽  
Florence Depardieu ◽  
Nancy Bourdon ◽  
Marguerite Fines-Guyon ◽  
Pierre Berger ◽  
...  
Keyword(s):  
Enterococcus Faecium ◽  
Constitutive Expression ◽  
Degenerate Primers ◽  
Glycopeptide Resistance ◽  
Content Type ◽  
Pcr Product ◽  
Low Levels ◽  
Rapid Amplification ◽  
Race Pcr ◽  
Tail Pcr

ABSTRACTEnterococcus faeciumUCN71, isolated from a blood culture, was resistant to low levels of vancomycin (MIC, 16 μg/ml) but susceptible to teicoplanin (MIC, 0.5 μg/ml). No amplification was observed with primers specific for the previously described glycopeptide resistance ligase genes, but a PCR product corresponding to a gene calledvanNwas obtained using degenerate primers and was sequenced. The deduced VanN protein was related (65% identity) to thed-alanine:d-serine VanL ligase. The organization of thevanNgene cluster, determined using degenerate primers and by thermal asymmetric interlaced (TAIL)-PCR, was similar to that of thevanCoperons. A single promoter upstream from the resistance operon was identified by rapid amplification of cDNA ends (RACE)-PCR. The presence of peptidoglycan precursors ending ind-serine andd,d-peptidase activities in the absence of vancomycin indicated constitutive expression of the resistance operon. VanN-type resistance was transferable by conjugation toE. faecium. This is the first report of transferabled-Ala-d-Ser-type resistance inE. faecium.

Hedgehog gene in Qingdao amphioxus Branchiostoma belcheri tsingtauense: cloning and expression pattern in early development

2002 ◽  
Vol 82 (4) ◽  
pp. 629-633 ◽  
Author(s):  
Y. Zhang ◽  
B. Mao ◽  
S. Zhang ◽  
H. Zhang
Keyword(s):  
Neural Tube ◽  
Cloning And Expression ◽  
Degenerate Primers ◽  
Reading Frame ◽  
Conserved Sequence ◽  
Branchiostoma Belcheri ◽  
Pcr Products ◽  
Rapid Amplification ◽  
Sonic Hedgehog Gene ◽  
High Level

A fragment of hedgehog was first isolated by polymerase chain reaction (PCR) from genomic DNA of Qingdao amphioxus using degenerate primers designed according to the conserved sequence in the second exon of hedgehog family. Then RT-PCR and rapid amplification of cDNA ends (3′ and 5′ RACE) were carried out using the mRNA of 18-h neurulae as template. The PCR products were sequenced and constituted a 3540 bp-long hedgehog gene, containing a complete open reading frame. The predicted protein is 415-amino-acids long, showing a high level of identity to other hedgehog proteins. The results show that Qingdao amphioxus contains one and probably only one hedgehog gene. Whole mount in situ hybridization revealed that hedgehog expressed in the prospective notochord, the notochord, floor plate of neural tube and mouth, similarly to its vertebrate homologue Sonic-hedgehog gene. This suggested that amphioxus hedgehog gene might function in the notochord induction to neural tube cells and in the development of the mouth.

scholarly journals Functional and molecular identification of a TASK-1 potassium channel regulating chloride secretion through CFTR channels in the shark rectal gland: implications for cystic fibrosis

2016 ◽  
Vol 311 (6) ◽  
pp. C884-C894 ◽  
Author(s):  
Connor J. Telles ◽  
Sarah E. Decker ◽  
William W. Motley ◽  
Alexander W. Peters ◽  
Ali Poyan Mehr ◽  
...  
Keyword(s):  
Potassium Channel ◽  
Reversal Potential ◽  
Chloride Secretion ◽  
Rectal Gland ◽  
Degenerate Primers ◽  
Reading Frame ◽  
Genomic Walking ◽  
Shark Rectal Gland ◽  
Mammalian Lung ◽  
Rapid Amplification

In the shark rectal gland (SRG), apical chloride secretion through CFTR channels is electrically coupled to a basolateral K+ conductance whose type and molecular identity are unknown. We performed studies in the perfused SRG with 17 K+ channel inhibitors to begin this search. Maximal chloride secretion was markedly inhibited by low-perfusate pH, bupivicaine, anandamide, zinc, quinidine, and quinine, consistent with the properties of an acid-sensitive, four-transmembrane, two-pore-domain K+ channel (4TM-K2P). Using PCR with degenerate primers to this family, we identified a TASK-1 fragment in shark rectal gland, brain, gill, and kidney. Using 5′ and 3′ rapid amplification of cDNA ends PCR and genomic walking, we cloned the full-length shark gene (1,282 bp), whose open reading frame encodes a protein of 375 amino acids that was 80% identical to the human TASK-1 protein. We expressed shark and human TASK-1 cRNA in Xenopus oocytes and characterized these channels using two-electrode voltage clamping. Both channels had identical current-voltage relationships (outward rectifying) and a reversal potential of −90 mV. Both were inhibited by quinine, bupivicaine, and acidic pH. The pKa for current inhibition was 7.75 for shark TASK-1 vs. 7.37 for human TASK-1, values similar to the arterial pH for each species. We identified this protein in SRG by Western blot and confocal immunofluorescent microscopy and detected the protein in SRG and human airway cells. Shark TASK-1 is the major K+ channel coupled to chloride secretion in the SRG, is the oldest 4TM 2P family member identified, and is the first TASK-1 channel identified to play a role in setting the driving force for chloride secretion in epithelia. The detection of this potassium channel in mammalian lung tissue has implications for human biology and disease.

An electrogenic Na+- HCO 3 − cotransporter (NBC) with a novel COOH-terminus, cloned from rat brain

2000 ◽  
Vol 278 (6) ◽  
pp. C1200-C1211 ◽  
Author(s):  
Mark O. Bevensee ◽  
Bernhard M. Schmitt ◽  
Inyeong Choi ◽  
Michael F. Romero ◽  
Walter F. Boron
Keyword(s):  
Rat Brain ◽  
Cortical Neurons ◽  
Polyclonal Antibodies ◽  
Amino Acid Level ◽  
Cdna Libraries ◽  
Human Pancreas ◽  
Opposite Pattern ◽  
Reading Frame ◽  
Expression Studies ◽  
Rapid Amplification

We screened rat brain cDNA libraries and used 5′ rapid amplification of cDNA ends to clone two electrogenic Na+-[Formula: see text] cotransporter (NBC) isoforms from rat brain (rb1NBC and rb2NBC). At the amino acid level, one clone (rb1NBC) is 96% identical to human pancreas NBC. The other clone (rb2NBC) is identical to rb1NBC except for 61 unique COOH-terminal amino acids, the result of a 97-bp deletion near the 3′ end of the open-reading frame. Using RT-PCR, we confirmed that mRNA from rat brain contains this 97-bp deletion. Furthermore, we generated rabbit polyclonal antibodies that distinguish between the unique COOH-termini of rb1NBC (αrb1NBC) and rb2NBC (αrb2NBC). αrb1NBC labels an ∼130-kDa protein predominantly from kidney, and αrb2NBC labels an ∼130-kDa protein predominantly from brain. αrb2NBC labels a protein that is more highly expressed in cortical neurons than astrocytes cultured from rat brain; αrb1NBC exhibits the opposite pattern. In expression studies, applying 1.5% CO2/10 mM [Formula: see text] to Xenopus oocytes injected with rb2NBC cRNA causes 1) pHi to recover from the initial CO2-induced acidification and 2) the cell to hyperpolarize. Subsequently, removing external Na+ reverses the pHi increase and elicits a rapid depolarization. In the presence of 450 μM DIDS, removing external Na+ has no effect on pHi and elicits a small hyperpolarization. The rate of the pHidecrease elicited by removing Na+ is insensitive to removing external Cl−. Thus rb2NBC is a DIDS-sensitive, electrogenic NBC that is predominantly expressed in brain of at least rat.

Cloning and functional characterization of an uncoupling protein homolog in hummingbirds

2001 ◽  
Vol 5 (3) ◽  
pp. 137-145 ◽  
Author(s):  
CLAUDIA R. VIANNA ◽  
THILO HAGEN ◽  
CHEN-YU ZHANG ◽  
ERIC BACHMAN ◽  
OLIVIER BOSS ◽  
...  
Keyword(s):  
Uncoupling Protein ◽  
Expression System ◽  
Functional Characterization ◽  
Pectoral Muscle ◽  
Mitochondrial Fraction ◽  
Mrna Levels ◽  
Coding Region ◽  
Reading Frame ◽  
Rapid Amplification

The cDNA of an uncoupling protein (UCP) homolog has been cloned from the swallow-tailed hummingbird, Eupetomena macroura. The hummingbird uncoupling protein (HmUCP) cDNA was amplified from pectoral muscle (flight muscle) using RT-PCR and primers for conserved domains of various known UCP homologs. The rapid amplification of cDNA ends (RACE) method was used to complete the cloning of the 5′ and 3′ ends of the open reading frame. The HmUCP coding region contains 915 nucleotides, and the deduced protein sequence consists of 304 amino acids, being ∼72, 70, and 55% identical to human UCP3, UCP2, and UCP1, respectively. The uncoupling activity of this novel protein was characterized in yeast. In this expression system, the 12CA5-tagged HmUCP fusion protein was detected by Western blot in the enriched mitochondrial fraction. Similarly to rat UCP1, HmUCP decreased the mitochondrial membrane potential as measured in whole yeast by uptake of the fluorescent potential-sensitive dye 3′,3-dihexyloxacarbocyanine iodide. The HmUCP mRNA is primarily expressed in skeletal muscle, but high levels can also be detected in heart and liver, as assessed by Northern blot analysis. Lowering the room’s temperature to 12–14°C triggered the cycle torpor/rewarming, typical of hummingbirds. Both in the pectoral muscle and heart, HmUCP mRNA levels were 1.5- to 3.4-fold higher during torpor. In conclusion, this is the first report of an UCP homolog in birds. The data indicate that HmUCP has the potential to function as an UCP and could play a thermogenic role during rewarming.

scholarly journals Lettuce Chlorosis Virus Disease: A New Threat to Cannabis Production

Viruses ◽  
2019 ◽  
Vol 11 (9) ◽  
pp. 802 ◽  
Author(s):  
Hadad ◽  
Luria ◽  
Smith ◽  
Sela ◽  
Lachman ◽  
...  
Keyword(s):  
Cannabis Sativa ◽  
Virus Disease ◽  
Middle Eastern ◽  
Sequencing Analysis ◽  
Disease Symptoms ◽  
Next Generation Sequencing Analysis ◽  
Nutrition Deprivation ◽  
Rapid Amplification ◽  
Race Pcr ◽  
Complete Viral Genome

In a survey conducted in Cannabis sativa L. (cannabis) authorized farms in Israel, plants showed disease symptoms characteristic of nutrition deprivation. Interveinal chlorosis, brittleness, and occasional necrosis were observed in older leaves. Next generation sequencing analysis of RNA extracted from symptomatic leaves revealed the presence of lettuce chlorosis virus (LCV), a crinivirus that belongs to the Closteroviridae family. The complete viral genome sequence was obtained using RT-PCR and Rapid Amplification of cDNA Ends (RACE) PCR followed by Sanger sequencing. The two LCV RNA genome segments shared 85–99% nucleotide sequence identity with LCV isolates from GenBank database. The whitefly Bemisia tabaci Middle Eastern Asia Minor1 (MEAM1) biotype transmitted the disease from symptomatic cannabis plants to un-infected ‘healthy’ cannabis, Lactuca sativa, and Catharanthus roseus plants. Shoots from symptomatic cannabis plants, used for plant propagation, constituted a primary inoculum of the disease. To the best of our knowledge, this is the first report of cannabis plant disease caused by LCV.

scholarly journals A variant of Runx2 that differs from the bone isoform in its splicing is expressed in spermatogenic cells

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e1862
Author(s):  
Satoru Kanto ◽  
Marcin Grynberg ◽  
Yoshiyuki Kaneko ◽  
Jun Fujita ◽  
Masanobu Satake
Keyword(s):  
Bone Development ◽  
Helical Conformation ◽  
Cdna Clones ◽  
Runt Domain ◽  
Reading Frame ◽  
Amino Terminal ◽  
Rapid Amplification ◽  
Differentiation Stage ◽  
Pachytene Spermatocytes ◽  
Immunohistochemical Analyses

Background.Members of theRunxgene family encode transcription factors that bind to DNA in a sequence-specific manner. Among the three Runx proteins, Runx2 comprises 607 amino acid (aa) residues, is expressed in bone, and plays crucial roles in osteoblast differentiation and bone development. We examined whether theRunx2gene is also expressed in testes.Methods.Murine testes from 1-, 2-, 3-, 4-, and 10-week-old male mice of the C57BL/6J strain andW∕Wvstrain were used throughout the study. Northern Blot Analyses were performed using extracts form the murine testes. Sequencing of cDNA clones and 5′-rapid amplification of cDNA ends were performed to determine the full length of the transcripts, which revealed that the testicular Runx2 comprises 106 aa residues coding novel protein. Generating an antiserum using the amino-terminal 15 aa of Runx2 (Met1to Gly15) as an antigen, immunoblot analyses were performed to detect the predicted polypeptide of 106 aa residues with the initiating Met1. With the affinity-purified anti-Runx2 antibody, immunohistochemical analyses were performed to elucidate the localization of the protein. Furthermore, bioinformatic analyses were performed to predict the function of the protein.Results.ARunx2transcript was detected in testes and was specifically expressed in germ cells. Determination of the transcript structure indicated that the testicularRunx2is a splice isoform. The predicted testicular Runx2 polypeptide is composed of only 106 aa residues, lacks a Runt domain, and appears to be a basic protein with a predominantly alpha-helical conformation. Immunoblot analyses with an anti-Runx2 antibody revealed that Met1in the deduced open reading frame ofRunx2is used as the initiation codon to express an 11 kDa protein. Furthermore, immunohistochemical analyses revealed that the Runx2 polypeptide was located in the nuclei, and was detected in spermatocytes at the stages of late pachytene, diplotene and second meiotic cells as well as in round spermatids. Bioinformatic analyses suggested that the testicular Runx2 is a histone-like protein.Discussion.A variant ofRunx2that differs from the bone isoform in its splicing is expressed in pachytene spermatocytes and round spermatids in testes, and encodes a histone-like, nuclear protein of 106 aa residues. Considering its nuclear localization and differentiation stage-dependent expression, Runx2 may function as a chromatin-remodeling factor during spermatogenesis. We thus conclude that a singleRunx2gene can encode two different types of nuclear proteins, a previously defined transcription factor in bone and cartilage and a short testicular variant that lacks a Runt domain.

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