scholarly journals A Cluster of Four Receptor-Like Genes Resides in the Vf Locus That Confers Resistance to Apple Scab Disease

Genetics ◽  
2002 ◽  
Vol 162 (4) ◽  
pp. 1995-2006
Author(s):  
Mingliang Xu ◽  
Schuyler S Korban
Keyword(s):  
Amino Acid ◽  
Expression Profiles ◽  
Venturia Inaequalis ◽  
Apple Scab ◽  
Amino Acid Sequences ◽  
Bac Contig ◽  
Mature Leaves ◽  
Rapid Amplification ◽  
Leucine Rich Repeats ◽  
High Degree

Abstract The Vf locus, derived from the crabapple species Malus floribunda 821, confers resistance to five races of the fungal pathogen Venturia inaequalis, the causal agent of apple scab disease. In our previous research, the Vf locus was restricted to a BAC contig of ∼290 kb covered by five overlapping BAC clones. Here, we report on cloning of the resistance gene(s) present in the Vf BAC contig using a highly reliable and straightforward approach. This approach relies on hybridization of labeled cDNAs to amplified inserts of subclones derived from BAC inserts, followed by recovery of full-size transcripts by rapid amplification of cDNA ends (RACE). A cluster of four resistance paralogs (Vfa1, Vfa2, Vfa3, and Vfa4) was identified in the Vf locus. Vfa1, Vfa2 and Vfa4 had no introns and are predicted to encode proteins characterized with extracellular leucine-rich repeats (LRRs) and transmembrane (TM) domains. However, Vfa3 contains an insertion of 780 bp at the end of the LRR motif, resulting in multiple truncated transcripts. Comparison of Vfa1, Vfa2, and Vfa4 paralogs revealed a high degree of overall homology in their deduced amino acid sequences, while divergences were mainly restricted within LRR domains, including variable LRR units, numerous amino acid substitutions, and several residue deletions/duplications. Differential expression profiles among the four paralogs were observed during leaf development. Vfa1, Vfa2, and Vfa3 were active in immature leaves, but slightly expressed in mature leaves, while Vfa4 was active in immature leaves and was highly expressed in mature leaves.

scholarly journals The Differential Binding Affinities of the Luteinizing Hormone (LH)/Choriogonadotropin Receptor for LH and Choriogonadotropin Are Dictated by Different Extracellular Domain Residues

2005 ◽  
Vol 19 (5) ◽  
pp. 1263-1276 ◽  
Author(s):  
Colette Galet ◽  
Mario Ascoli
Keyword(s):  
Amino Acid ◽  
Ligand Binding ◽  
Binding Affinity ◽  
Extracellular Domain ◽  
Binding Affinities ◽  
Amino Acid Residues ◽  
Identify Amino Acid ◽  
Β Sheets ◽  
Leucine Rich Repeats ◽  
High Degree

Abstract The high degree of amino acid sequence homology and the divergent ligand binding affinities of the rat (r) and human (h) LH receptors (LHRs) allowed us to identify amino acid residues of their extracellular domain that are responsible for the different binding affinities of bovine (b) and hLH, and human choriogonadotropin (hCG) to the hLHR and rLHR. Because of the proposed importance of the β-sheets of the leucine-rich repeats (LRRs) of the extracellular domain of the LHR on hormone binding, we examined 10 divergent residues present in these regions by analyzing two complementary sets of mutants in which hLHR residues were substituted with the corresponding rLHR residues and vice versa. These experiments resulted in the identification of a single residue (a Ile or Ser in the C-terminal end of LRR2 of the hLHR or rLHR, respectively) that is important for hLH binding affinity. Surprisingly, however, this residue does not affect hCG or for bLH binding affinity. In fact, the results obtained with bLH and hCG show that several of the divergent residues in the β-sheets of LRR1–9 affect bLH binding affinity, but none of them affect hCG binding affinity. Importantly, our results also emphasize the involvement of residues outside of the β-sheets of the LRRs of the LHR in ligand binding affinity. This finding has to be considered in future models of the interaction of LH/CG with the LHR.

Carnivora: The Amino Acid Sequence of the Adult European Polecat (Mustela putorius, Mustelidae) Hemoglobins

1989 ◽  
Vol 44 (7) ◽  
pp. 817-824 ◽  
Author(s):  
Aftab Ahmed ◽  
Meeno Jahan ◽  
Gerhard Braunitzer ◽  
Helmut Pechlaner
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Gas Phase ◽  
Primary Structure ◽  
Amino Acid Sequences ◽  
Mustela Putorius ◽  
Globin Chains ◽  
The Family ◽  
High Degree ◽  
Β Chain

The complete amino acid sequences of the hemoglobins from the adult European polecat (Mustela putorius) are presented. The erythrocytes contain two hemoglobin components and three globin chains (α I, α II and β). The primary structure of globin chains and of the tryptic peptides determined in liquid- and gas-phase sequantors. Comparing the sequences of the globin chains of the polecat with that of human Hb-A, 17 (23.9%) substitutions were recognized in the α I, 16 (22.5%) in the α II and 14 (20.4%) in the β chain. A high degree of homology observed with other representatives of the family Mustelidae.

Molecular cloning, characterization and tissue specificity of the expression in the TAC1 genes from Henan Huai goat (Capra hircus)

2019 ◽  
Author(s):  
Zhilong Tian ◽  
Yuqin Wang ◽  
Huibin Shi ◽  
Zhibo Wu ◽  
Xiaohui Zhang ◽  
...  
Keyword(s):  
Amino Acid ◽  
Phylogenetic Analyses ◽  
Expression Patterns ◽  
Amino Acid Sequences ◽  
Full Length ◽  
Capra Hircus ◽  
Reading Frame ◽  
Relative Molecular Weight ◽  
Rapid Amplification ◽  
And Function

To further to understand the structure and function of the TAC1 gene, we cloned the full-length cDNAs of the TAC1 genes from goat by rapid amplification of cDNA ends-PCR and the qRT-PCR was used to analyze the TAC1 mRNA expression patterns of goat various tissues. The full-length cDNA of goat TAC1 was 1176 bp, with a 339 bp open reading frame encoding 112 amino acids. The amino acid sequence analysis revealed that goat TAC1 gene encoded a water-drain protein and its relative molecular weight and isoelectric point was 13,012.86 Da and 6.29 respectively. Alignment and phylogenetic analyses revealed that their amino acid sequences were highly similar to those of other vertebrates. TAC1 expression of the goat of the brain, cerebellum, medulla oblongata, heart, liver, spleen, lung, kidney, uterus, ovaries. These results serve as a foundation for further study on the Capra hircus TAC1 gene.

scholarly journals The Rvi15 (Vr2) Apple Scab Resistance Locus Contains Three TIR-NBS-LRR Genes

2010 ◽  
Vol 23 (5) ◽  
pp. 608-617 ◽  
Author(s):  
Paolo Galli ◽  
Andrea Patocchi ◽  
Giovanni Antonio Lodovico Broggini ◽  
Cesare Gessler
Keyword(s):  
Venturia Inaequalis ◽  
Interleukin 1 ◽  
Apple Scab ◽  
Scab Resistance ◽  
Receptor Protein ◽  
Resistance Locus ◽  
Fresh Market ◽  
High Quality ◽  
Bac Clone ◽  
Bac Contig

Scab caused by the pathogen Venturia inaequalis is considered the most important fungal disease of cultivated apple (Malus × domestica Borkh.). In all, 16 monogenic resistances against scab have been found in different Malus spp. and some of them are currently used in apple breeding for scab-resistant cultivars. However, the self incompatibility and the long generation time of Malus spp. together with the high standards of fruit quality demanded from the fresh market render the breeding of high-quality cultivars in apple a long and expensive task. Therefore, the cloning of disease resistance genes and the use of the cloned genes for the transformation of high-quality apple cultivars could be an approach to solve these drawbacks. We report the construction of a bacterial artificial chromosome (BAC) contig spanning the Rvi15 (Vr2) apple scab resistance locus using two GMAL 2473 BAC libraries. A single BAC clone of the contig was sufficient to span the resistance locus. The BAC clone was completely sequenced, allowing identification of a sequence of 48.6 kb going from the two closest markers (ARGH17 and 77G20RP) bracketing Rvi15 (Vr2). Analysis of the 48.6-kb sequence revealed the presence of three putative genes characterized by a Toll and mammalian interleukin-1 receptor protein nucleotide-binding site leucine-rich repeat structure. All three genes were found to be transcribed.

scholarly journals Purification and Characterization of AsES Protein

2013 ◽  
Vol 288 (20) ◽  
pp. 14098-14113 ◽  
Author(s):  
Nadia R. Chalfoun ◽  
Carlos F. Grellet-Bournonville ◽  
Martín G. Martínez-Zamora ◽  
Araceli Díaz-Perales ◽  
Atilio P. Castagnaro ◽  
...  
Keyword(s):  
Amino Acid ◽  
Proteolytic Activity ◽  
Foliar Spray ◽  
Amino Acid Sequences ◽  
Purification And Characterization ◽  
Degenerate Primers ◽  
Acremonium Strictum ◽  
Rapid Amplification

In this work, the purification and characterization of an extracellular elicitor protein, designated AsES, produced by an avirulent isolate of the strawberry pathogen Acremonium strictum, are reported. The defense eliciting activity present in culture filtrates was recovered and purified by ultrafiltration (cutoff, 30 kDa), anionic exchange (Q-Sepharose, pH 7.5), and hydrophobic interaction (phenyl-Sepharose) chromatographies. Two-dimensional SDS-PAGE of the purified active fraction revealed a single spot of 34 kDa and pI 8.8. HPLC (C2/C18) and MS/MS analysis confirmed purification to homogeneity. Foliar spray with AsES provided a total systemic protection against anthracnose disease in strawberry, accompanied by the expression of defense-related genes (i.e. PR1 and Chi2-1). Accumulation of reactive oxygen species (e.g. H2O2 and O2̇̄) and callose was also observed in Arabidopsis. By using degenerate primers designed from the partial amino acid sequences and rapid amplification reactions of cDNA ends, the complete AsES-coding cDNA of 1167 nucleotides was obtained. The deduced amino acid sequence showed significant identity with fungal serine proteinases of the subtilisin family, indicating that AsES is synthesized as a larger precursor containing a 15-residue secretory signal peptide and a 90-residue peptidase inhibitor I9 domain in addition to the 283-residue mature protein. AsES exhibited proteolytic activity in vitro, and its resistance eliciting activity was eliminated when inhibited with PMSF, suggesting that its proteolytic activity is required to induce the defense response. This is, to our knowledge, the first report of a fungal subtilisin that shows eliciting activity in plants. This finding could contribute to develop disease biocontrol strategies in plants by activating its innate immunity.

scholarly journals Red Clover Hydroxycinnamoyl-CoA:L-DOPA Hydroxycinnamoyl Transferase, a BAHD Hydroxycinnamoyl-Coenzyme A:L-3,4-Dihydroxyphenylalanine Hydroxycinnamoyl Transferase That Synthesizes Clovamide and Other N-Hydroxycinnamoyl-Aromatic Amino Acid Amides

2021 ◽  
Vol 12 ◽  
Author(s):  
Michael L. Sullivan ◽  
Benjamin J. Knollenberg
Keyword(s):  
Amino Acid ◽  
Rational Design ◽  
Aromatic Amino Acids ◽  
Red Clover ◽  
In Planta ◽  
Degenerate Primers ◽  
Coa Transferase ◽  
Rapid Amplification ◽  
Coa Thioesters ◽  
High Degree

Red clover leaves accumulate high levels (up to 1 to 2% of dry matter) of two caffeic acid derivatives: phaselic acid (2-O-caffeoyl-L-malate) and clovamide [N-caffeoyl-L-3,4-dihydroxyphenylalanine (L-DOPA)]. These likely play roles in protecting the plant from biotic and abiotic stresses but can also help preserve protein during harvest and storage of the forage via oxidation by an endogenous polyphenol oxidase. We previously identified and characterized, a hydroxycinnamoyl-coenzyme A (CoA):malate hydroxycinnamoyl transferase (HMT) from red clover. Here, we identified a hydroxycinnamoyl-CoA:L-DOPA hydroxycinnamoyl transferase (HDT) activity in unexpanded red clover leaves. Silencing of the previously cloned HMT gene reduced both HMT and HDT activities in red clover, even though the HMT enzyme lacks HDT activity. A combination of PCR with degenerate primers based on BAHD hydroxycinnamoyl-CoA transferase sequences and 5′ and 3′ rapid amplification of cDNA ends was used to clone two nearly identical cDNAs from red clover. When expressed in Escherichia coli, the encoded proteins were capable of transferring hydroxycinnamic acids (p-coumaric, caffeic, or ferulic) from the corresponding CoA thioesters to the aromatic amino acids L-Phe, L-Tyr, L-DOPA, or L-Trp. Kinetic parameters for these substrates were determined. Stable expression of HDT in transgenic alfalfa resulted in foliar accumulation of p-coumaroyl- and feruloyl-L-Tyr that are not normally present in alfalfa, but not derivatives containing caffeoyl or L-DOPA moieties. Transient expression of HDT in Nicotiana benthamiana resulted in the production of caffeoyl-L-Tyr, but not clovamide. Coexpression of HDT with a tyrosine hydroxylase resulted in clovamide accumulation, indicating the host species’ pool of available amino acid (and hydroxycinnamoyl-CoA) substrates likely plays a major role in determining HDT product accumulation in planta. Finally, that HDT and HMT proteins share a high degree of identity (72%), but differ substantially in substrate specificity, is promising for further investigation of structure-function relationships of this class of enzymes, which could allow the rational design of BAHD enzymes with specific and desirable activities.

Identification and Complete Genome Sequence of an Alternavirus From Airborne Spores of the Phytopathogenic Fungus Fusarium Bullatum

2021 ◽  
Author(s):  
Samira Mokhtari ◽  
Akhtar Ali
Keyword(s):  
Phylogenetic Analysis ◽  
Amino Acid ◽  
Amino Acid Sequences ◽  
Phytopathogenic Fungus ◽  
Sequencing Analysis ◽  
Airborne Spores ◽  
Double Stranded Rna ◽  
Rapid Amplification ◽  
Dsrna Mycovirus ◽  
Blastn Search

Abstract A double-stranded RNA (dsRNA) mycovirus was isolated from airborne spores of Fusarium bullatum and was named Fusarium bullatum alternavirus 1 (FbAV1). Sequencing analysis and the rapid amplification of cDNA ends (RACE) of 5’ and 3’-end confirmed three segments: dsRNA1 (3546 nt), dsRNA2 (2511 nt) and dsRNA3 (2484 nt). BLASTN search of sequences showed that FbAV1 has 92-96% identity with Fusarium incarnatum Alternavirus 1 (FiAV1). Phylogenetic analysis of the RdRp amino acid sequences suggested that the dsRNA mycovirus in this study clustered with the newly proposed family “Alternaviridae”. This is the first report of FbAV1 mycovirus from airborne spores of a fungus F. bullatum.

Computational Characterisation and Identification of Peptides for in silico Detection of Potentially Celiac-Toxic Proteins

2007 ◽  
Vol 13 (2) ◽  
pp. 125-133 ◽  
Author(s):  
M. Darewicz ◽  
J. Dziuba ◽  
P. Minkiewicz
Keyword(s):  
Celiac Disease ◽  
Amino Acid ◽  
In Silico ◽  
In Silico Analysis ◽  
Amino Acid Sequences ◽  
Random Coil ◽  
Sequence Motifs ◽  
Peptide Epitopes ◽  
High Degree ◽  
Toxic Peptides

This work reports on in silico analysis of celiac-toxic peptide occurrence in proteins. The toxic properties of celiac disease are linked to the presence of specific amino acid sequences and the properties of their environment. The analysed celiac-toxic peptides were found to be predominated by unordered structures of random coil and β-turns. Proline and glutamine-rich amino acid sequences from hydrophilic β-turns were exposed on the surface of the precursor proteins. The sequence motifs represented by gluten peptide epitopes or tetrapeptides with surroundings seem to represent an immunodominant structure. The application of MS BLAST software enabled identification of a few fragments with high degrees of identity to the toxic peptides in one protein sequence. Rich sources of celiac-disease-potentiating peptides were wheat gliadins, barley hordeins and rye secalins as well as low-molecular weight fractions of glutenin. In addition, amino acid sequences with a high degree of identity to the toxic peptides examined were detected in maize zein, oat avenin, protein of rice, yeast and chicken muscles, as well as β-casein and galanin.

scholarly journals Affinity purification of 5-methylthioribose kinase and 5-methylthioadenosine/S-adenosylhomocysteine nucleosidase from Klebsiella pneumoniae

1996 ◽  
Vol 317 (1) ◽  
pp. 285-290 ◽  
Author(s):  
Kenneth A. CORNELL ◽  
R. W. WINTER ◽  
Paula A. TOWER ◽  
Michael K. RISCOE
Keyword(s):  
Amino Acid ◽  
Molecular Mass ◽  
Sequence Similarity ◽  
Affinity Purification ◽  
Amino Acid Sequences ◽  
Reading Frame ◽  
Sds Page ◽  
High Degree ◽  
Putative Translation Product ◽  
Substrate Kinetics

Two enzymes in the methionine salvage pathway, 5-methylthioribose kinase (MTR kinase) and 5´-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTA/SAH nucleosidase) were purified from Klebsiellapneumoniae. Chromatography using a novel 5´-(p-aminophenyl)thioadenosine/5-(p-aminophenyl)thioribose affinity matrix allowed the binding and selective elution of each of the enzymes in pure form. The molecular mass, substrate kinetics and N-terminal amino acid sequences were characterized for each of the enzymes. Purified MTR kinase exhibits an apparent molecular mass of 46–50 kDa by SDS/PAGE and S200HR chromatography, and has a Km for MTR of 12.2 μM. Homogeneous MTA/SAH nucleosidase displays a molecular mass of 26.5 kDa by SDS/PAGE, and a Km for MTA of 8.7 μM. Comparisons of the N-terminal sequences obtained for each of the enzymes with protein-sequence databases failed to reveal any significant sequence similarities to known proteins. However, the amino acid sequence obtained for the nucleosidase did share a high degree of sequence similarity with the putative translation product of an open reading frame in Escherichia coli, thus providing a tentative identification of this gene as encoding an MTA/SAH nucleosidase.

scholarly journals Recombinant production of rabbit β-Nerve Growth Factor and its biological effect on rabbit sperm

2018 ◽  
Author(s):  
Ana Sanchez-Rodriguez ◽  
Paloma Abad ◽  
Maria Arias-Alvarez ◽  
Pilar G. Rebollar ◽  
José M. Bautista ◽  
...  
Keyword(s):  
Amino Acid ◽  
Nerve Growth Factor ◽  
Growth Factor ◽  
Recombinant Protein ◽  
Dose Response ◽  
Amino Acid Sequences ◽  
Nerve Growth ◽  
Specificity Effect ◽  
Rapid Amplification ◽  
Dose Response Effect

AbstractThe neurotrophin β-Nerve Growth Factor (β-NGF) is flourishing as a protein with important roles in the ovulation induction process in induced-ovulation species but data in rabbits are still inconclusive, probably due to the species-specificity effect of the neurotrophin to trigger the ovulation. Moreover, β-NGF seems to have a role in sperm function. To clarify these functionalities we aimed, in the present research: 1) to newly synthesize a functional recombinant β-NGF from rabbit (rrβ-NGF), 2) to reveal differences in the amino acid sequence of rabbit β-NGF compared to other sequences of induced and spontaneous ovulator species, and 3) to assess the effects of rrβ-NGF on sperm viability and motility. The nucleotide sequence of NGF from rabbit prostate was sequenced by Rapid Amplification of cDNA Ends (RACE) and annotated in GenBank (KX528686). Then, rrβ-NGF was produced in CHO cells and purified by affinity chromatography. Western blot and MALDI-TOF analyses confirmed the correct identity of the recombinant protein. rrβ-NGF functionality was validated in PC12 cells through a successful dose-response effect along 8 days. The comparison of the amino acid sequences of NGF between rabbit and other species suggested some relevant substitutions at its binding site to both the high-(TrkA) and the low-(p75) affinity receptors. The addition of rrβ-NGF in rabbit sperm, in a time- and dose-response study, did not affect its viability but slightly changed some of its motility parameters at the highest concentration used (100 ng/ml). Thus, it can be considered that this new recombinant protein may be used for biotechnological and reproduction assisted techniques in ovulation-induced species.

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