Polymorphism of genes controlling low level of linolenic acid in lines from VIR flax genetic collection

Background. Linseed solin varieties were created for nutrition, but the effect of oil fatty acid (FA) composition on other characters is not clear. Materials and methods. Using 6 inbreeding generations from 26 heterogeneous flax accessions were generated 19 high (HL), 7 medium (ML) and 14 low linolenic (LL) lines. For each lines contents of 5 basic FA: palmitic, stearic, oleic (OLE), linoleic (LIO) and linolenic (LIN); the ratio LIO/LIN, oil iodine number, vegetative period (VP) phases and plants size were evaluated. Development of CAPS marker for LuFAD3A gene was performed using idtdna.com. Sequencing of LIN genes sites was done in the Centre MCT SPBGU and Eurogen. Results. ANOVA showed significant differences HL, ML and LL groups for PAL, OLE, LIO, LIN, LIO/LIN, IOD. Considerable decrease of LIN, causes asymmetric changes in FA ratio and correlations between them and other traits. Factor analysis revealed the influence of two factors. The first one divided lines according to their LIN level and characters associated with it, the second one according to the VP and OLE. LIN synthesis is controlled by two complementary genes LuFAD3A and LuFAD3B. Sequencing of LuFAD3A gene 1 exon of 6 lines revealed a mutation (G255 A255), resulting in formation of stop codon. Developed developed CAPS-marker confirmed the homozygosity of hybrids between LL (gc-391) and HL lines (gc-65, 109, 121). Descendants of hybrid between gc-109 and gc-391 ripened 8-10 days earlier than gc-391. CAPS markers of LuFAD3B gene revealed differences between HL, ML, LL lines. Sequencing of this gene first exon and the beginning of the second one in 3 lines (1HL, 2LL) showed that this method reveals a mutation in the second restriction site, located in the 2 exon (C6 T6), and causing the replacement Hys Tyr. Conclusion. Lines from GC have wide variability of FA and other agronomic characters, combination of which will expand the cultivation of solin.

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Modification of recombinant human epidermal growth factor (rh-EGF) expression vector by site-directed mutagenesis for therapeutic protein production

Indonesian Journal of Biotechnology ◽

10.22146/ijbiotech.41859 ◽

2019 ◽

Vol 24 (1) ◽

pp. 8

Author(s):

Achmad Rodiansyah ◽

Riyona Desvy Pratiwi ◽

Sabighoh Zanjabila ◽

Asrul Muhamad Fuad

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Expression Vector ◽

Restriction Site ◽

Stop Codon ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Human Epidermal Growth Factor ◽

Epidermal Growth ◽

Ecori Restriction

Recombinant human epidermal growth factor (rh-EGF) has high value in therapies for h-EGF deficiency-related diseases. The expression of the h-EGF gene was designed by using the pET21b(+) vector and Escherichia coli BL21(DE3) as the expression host. In a previous study, the sequence of a 6xHis tag without any restriction sites was fused to the h-EGF gene, yet it was not possible to obtain a purified and single rh-EGF by this approach. In this study, we modified the rh-EGF expression vector using site-directed mutagenesis (SDM) to remove the sequence of the 6xHis tag. The vector modification was carried out by inserting a stop codon and the EcoRI restriction site, along with deleting the 6xHis tag sequence. The results of PCR showed non-specific bands, while 2-step cycles PCR produced one non-specific band, and 3-step cycles PCR produced two non-specific bands. After purification of the PCR products, the SDM-recombinant plasmids treated for template plasmid-free product were transformed into E. coli DH5a. Even though the transformation efficiency was low, the planned gene mutations including the deletion of the 6xHis tag and insertion of the stop codon and EcoRI restriction site in plasmid pET21b(+) were successfully carried out. When using this modified vector in expression studies, rh-EGF of a similar size to that of the rh-EGF standard and approximately 1 kDa smaller than the rh-EGF-6xHis of the previous study was obtained.

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Transcript profiling and gene characterization of three fatty acid desaturase genes in high, moderate, and low linolenic acid genotypes of flax (Linum usitatissimum L.) and their role in linolenic acid accumulation

Genome ◽

10.1139/g11-013 ◽

2011 ◽

Vol 54 (6) ◽

pp. 471-483 ◽

Cited By ~ 24

Author(s):

Mitali Banik ◽

Scott Duguid ◽

Sylvie Cloutier

Keyword(s):

Fatty Acid ◽

Linolenic Acid ◽

Seed Development ◽

Stop Codon ◽

Expression Patterns ◽

Fatty Acid Desaturase ◽

The Novel ◽

Transcript Accumulation ◽

Acid Accumulation ◽

Low Linolenic

Three genes encoding fatty acid desaturase 3 (fad3a, fad3b, and a novel fad3c) were cloned from four flax genotypes varying in linolenic acid content. Real-time PCR was used to quantify expression levels of the three fad3 genes during seed development. High amounts of both fad3a and fad3b transcripts were observed and reached their peak levels at 20 days after anthesis, except for fad3a from SP2047 where only low level expression was observed throughout seed development. Transcript accumulation of the novel fad3c gene was at similar background levels. The fatty acid composition was analysed for all genotypes and stages of development and compared with the fad3 gene expression patterns. α-Linolenic acid gradually accumulated during seed development, while linoleic acid was transient and decreased in M5791, UGG5-5, and AC McDuff. In contrast, the linolenic acid present in the early stages of development nearly completely disappeared in SP2047, while linoleic acid steadily accumulated. fad3a of the low linolenic acid line SP2047 encoded a truncated protein caused by a premature stop codon resulting from a single point mutation, and the low level of transcript accumulation in this genotype is likely due to nonsense-mediated mRNA decay caused by the premature termination of translation as a result of this early stop codon. Although substantial amounts of transcript accumulation occurred with fad3b of SP2047 genotype, cloning of the gene revealed a mutation in the first histidine box causing an amino acid change. Heterologous expression in yeast of the SP2047 and UGG5-5 fad3b genes showed that the mutation in the histidine box in SP2047 caused the enzyme inactivity. Taken together, these results showed that fad3a and fad3b are responsible for linolenic acid accumulation in flax seeds but did not support a major role for the novel fad3c. These observations were further supported by phenotypic and genotypic assessment of a doubled haploid population. Expression patterns of fad3a and fad3b were highly correlated with linolenic acid accumulation during seed development, with the exception of fad3b in SP2047 whose lack of activity was caused by the histidine box mutation despite its transcript accumulation being similar to that of the fad3b of the other genotypes.

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Molecular Study of Glanzmann Thrombasthenia in 3 Patients Issued from 2 Different Families

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649830 ◽

1995 ◽

Vol 74 (03) ◽

pp. 822-827 ◽

Cited By ~ 5

Author(s):

C Vinciguerra ◽

M C Trzeciak ◽

N Philippe ◽

D Frappaz ◽

J Reynaud ◽

...

Keyword(s):

Nonsense Mutation ◽

Restriction Site ◽

Stop Codon ◽

Hot Spot ◽

Genetic Defect ◽

Molecular Study ◽

Type I ◽

Glanzmann Thrombasthenia ◽

Cg Dinucleotide ◽

Homozygous Nonsense Mutation

SummaryIn an effort to further understand Glanzmann thrombasthenia (GT) 3 patients from 2 different families were studied. After biochemical and immunological analysis these patients were classified as type I. We observed in the first family a new restriction site for Stu I in exon II of the glycoprotein (GP) Ilia gene caused by a homozygous nonsense mutation: 62 Arg to stop codon. The parents were heterozygotes for this mutation. We found in the second family a previously described nonsense mutation: 584 Arg to stop codon in exon 17 of the GPIIb gene. The father and his two affected sons were heterozygous for this genetic defect. This mutation 62 Arg to stop codon is a new description of a genetic defect associated with GT. Furthermore, the discovery of the same mutation in 3 affected families from different ethnic groups raises the possibility of either a hot spot mutation in the CG dinucleotide region of GPIIb gene, or an ancient mutant allele present in diffuse populations at a relatively high frequency

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Conundrum of the Lack of Defective RNAs (dRNAs) Associated with Tobamovirus Infections: dRNAs That Can Move Are Not Replicated by the Wild-Type Virus; dRNAs That Are Replicated by the Wild-Type Virus Do Not Move

Journal of Virology ◽

10.1128/jvi.75.12.5518-5525.2001 ◽

2001 ◽

Vol 75 (12) ◽

pp. 5518-5525 ◽

Cited By ~ 25

Author(s):

Elisabeth Knapp ◽

William O. Dawson ◽

Dennis J. Lewandowski

Keyword(s):

De Novo ◽

Stop Codon ◽

Wild Type Virus ◽

Type Virus ◽

Wild Type ◽

In Planta ◽

Reading Frame ◽

Defective Rnas ◽

Two Factors ◽

Associated Proteins

ABSTRACT Two classes of artificially constructed defective RNAs (dRNAs) ofTobacco mosaic virus (TMV) were examined in planta with helper viruses that expressed one (183 kDa) or both (126 and 183 kDa) of the replicase-associated proteins. The first class of artificially constructed dRNAs had the helicase and polymerase (POL) domains deleted; the second had an intact 126-kDa protein open reading frame (ORF). Despite extremely high levels of replication in protoplasts, the first class of dRNAs did not accumulate in plants. The dRNAs with an intact 126-kDa protein ORF were replicated at moderate levels in protoplasts and in planta when supported by a TMV mutant that expressed the 183-kDa protein but not the 126-kDa protein (183F). These dRNAs were not supported by helper viruses expressing both replicase-associated proteins. De novo dRNAs were generated in plants infected by 183F but not in plants infected with virus with the wild-type replicase. These novel dRNAs each contained a new stop codon near the location of the wild-type stop codon for the 126-kDa protein and had most of the POL domain deleted. The fact that only dRNAs that contained a complete 126-kDa protein ORF moved systemically suggests that expression of a functional 126-kDa protein or the presence of certain sequences and/or structures within this ORF is required for movement of dRNAs. At least two factors may contribute to the lack of naturally occurring dRNAs in association with wild-type TMV infections: an inability of TMV to support dRNAs that can move in plants and the inability of dRNAs that can be replicated by TMV to move in plants.

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Nuclear Assembly of UGA Decoding Complexes on Selenoprotein mRNAs: a Mechanism for Eluding Nonsense-Mediated Decay?

Molecular and Cellular Biology ◽

10.1128/mcb.26.5.1795-1805.2006 ◽

2006 ◽

Vol 26 (5) ◽

pp. 1795-1805 ◽

Cited By ~ 39

Author(s):

Lucia A. de Jesus ◽

Peter R. Hoffmann ◽

Tanya Michaud ◽

Erin P. Forry ◽

Andrea Small-Howard ◽

...

Keyword(s):

Mammalian Cells ◽

Stop Codon ◽

Elongation Factor ◽

Protein Translation ◽

Nonsense Mediated Decay ◽

Nuclear Assembly ◽

Mrna Binding Protein ◽

Two Factors ◽

Mrna Binding ◽

Selenoprotein Mrnas

ABSTRACT Recoding of UGA from a stop codon to selenocysteine poses a dilemma for the protein translation machinery. In eukaryotes, two factors that are crucial to this recoding process are the mRNA binding protein of the Sec insertion sequence, SBP2, and the specialized elongation factor, EFsec. We sought to determine the subcellular localization of these selenoprotein synthesis factors in mammalian cells and thus gain insight into how selenoprotein mRNAs might circumvent nonsense-mediated decay. Intriguingly, both EFsec and SBP2 localization differed depending on the cell line but significant colocalization of the two proteins was observed in cells where SBP2 levels were detectable. We identify functional nuclear localization and export signals in both proteins, demonstrate that SBP2 undergoes nucleocytoplasmic shuttling, and provide evidence that SBP2 levels and localization may influence EFsec localization. Our results suggest a mechanism for the nuclear assembly of the selenocysteine incorporation machinery that could allow selenoprotein mRNAs to circumvent nonsense-mediated decay, thus providing new insights into the mechanism of selenoprotein translation.

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Conversion of a diversity arrays technology marker differentiating wild and cultivated carrots to a co-dominant cleaved amplified polymorphic site marker.

Acta Biochimica Polonica ◽

10.18388/abp.2014_1917 ◽

2014 ◽

Vol 61 (1) ◽

Cited By ~ 5

Author(s):

Alicja Macko-Podgórni ◽

Massimo Iorizzo ◽

Krzysztof Smółka ◽

Philipp W Simon ◽

Dariusz Grzebelus

Keyword(s):

Genetic Basis ◽

Restriction Site ◽

Polymorphic Site ◽

Caps Marker ◽

Specific Primers ◽

Diversity Arrays Technology ◽

Wild Carrot ◽

Pcr Products ◽

In The Wild ◽

Site Marker

Cultivated carrot and its wild ancestor co-occur in most temperate regions of the world and can easily hybridize. The genetic basis of the process of domestication in carrot is not well understood. Recent results of an investigation on genetic diversity structure of cultivated and wild carrot and signatures for domestication using Diversity Arrays Technology (DArT) allowed identification of polymorphisms differentiating wild and cultivated accessions. We selected one of these polymorphisms, showing the strongest evidence for directional selection in the course of domestication, and converted it into a co-dominant cleaved amplified polymorphic site (CAPS) marker named cult. To achieve that, we designed site-specific primers anchored in sequences flanking the original DArT clone, amplified and sequenced the PCR products derived from cultivated and wild carrot. A PstI restriction site present in the 'cultivated' variant and absent in the 'wild' was subsequently used for routine differentiation the two variants. We validated the cult marker on 88 accessions of cultivated and wild carrot, each represented by five individuals. The allelic variant associated with the wild phenotype was only rarely observed in cultivated carrot, mostly in purple-rooted accessions originating Turkey and Iran, possibly indicating that the physical association between the diagnostic polymorphism and the putative 'domestication gene' has been broken in a group of Eastern carrots.

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Slow Down You’re Moving Too Fast

Journal of Media Psychology Theories Methods and Applications ◽

10.1027/1864-1105/a000130 ◽

2015 ◽

Vol 27 (2) ◽

pp. 53-63 ◽

Cited By ~ 3

Author(s):

Annie Lang ◽

Nancy Schwartz ◽

Sharon Mayell

Keyword(s):

Older Adults ◽

Cognitive Aging ◽

Attention And Memory ◽

Younger Adults ◽

Media Messages ◽

Two Factors ◽

The Difference

The study reported here compared how younger and older adults processed the same set of media messages which were selected to vary on two factors, arousing content and valence. Results showed that older and younger adults had similar arousal responses but different patterns of attention and memory. Older adults paid more attention to all messages than did younger adults. However, this attention did not translate into greater memory. Older and younger adults had similar levels of memory for slow-paced messages, but younger adults outperformed older adults significantly as pacing increased, and the difference was larger for arousing compared with calm messages. The differences found are in line with predictions made based on the cognitive-aging literature.

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Effects of an Anxiety-Specific Psychometric Factor on Fear Conditioning and Fear Generalization

Zeitschrift für Psychologie ◽

10.1027/2151-2604/a000304 ◽

2017 ◽

Vol 225 (3) ◽

pp. 200-213 ◽

Cited By ~ 1

Author(s):

Christian Baumann ◽

Miriam A. Schiele ◽

Martin J. Herrmann ◽

Tina B. Lonsdorf ◽

Peter Zwanzger ◽

...

Keyword(s):

Risk Factor ◽

Anxiety Disorders ◽

Fear Conditioning ◽

Longitudinal Design ◽

Principal Component ◽

Anxiety And Depression ◽

Specific Factor ◽

High Anxious ◽

Two Factors ◽

Fear Generalization

Abstract. Conditioning and generalization of fear are assumed to play central roles in the pathogenesis of anxiety disorders. Here we investigate the influence of a psychometric anxiety-specific factor on these two processes, thus try to identify a potential risk factor for the development of anxiety disorders. To this end, 126 healthy participants were examined with questionnaires assessing symptoms of anxiety and depression and with a fear conditioning and generalization paradigm. A principal component analysis of the questionnaire data identified two factors representing the constructs anxiety and depression. Variations in fear conditioning and fear generalization were solely associated with the anxiety factor characterized by anxiety sensitivity and agoraphobic cognitions; high-anxious individuals exhibited stronger fear responses (arousal) during conditioning and stronger generalization effects for valence and UCS-expectancy ratings. Thus, the revealed psychometric factor “anxiety” was associated with enhanced fear generalization, an assumed risk factor for anxiety disorders. These results ask for replication with a longitudinal design allowing to examine their predictive validity.

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