Academic literature on the topic 'High-yield protein expression'
Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles
Consult the lists of relevant articles, books, theses, conference reports, and other scholarly sources on the topic 'High-yield protein expression.'
Next to every source in the list of references, there is an 'Add to bibliography' button. Press on it, and we will generate automatically the bibliographic reference to the chosen work in the citation style you need: APA, MLA, Harvard, Chicago, Vancouver, etc.
You can also download the full text of the academic publication as pdf and read online its abstract whenever available in the metadata.
Journal articles on the topic "High-yield protein expression"
1
Vasseur-Godbillon, Corinne, Djemel Hamdane, Michael C. Marden та Véronique Baudin-Creuza. "High-yield expression in Escherichia coli of soluble human α-hemoglobin complexed with its molecular chaperone". Protein Engineering, Design and Selection 19, № 3 (2006): 91–97. http://dx.doi.org/10.1093/protein/gzj006.
Full text
2
Lin, Bo, Hailing Meng, Hui Bing, Dongting Zhangsun, and Sulan Luo. "Efficient Expression of Acetylcholine-Binding Protein fromAplysia californicain Bac-to-Bac System." BioMed Research International 2014 (2014): 1–9. http://dx.doi.org/10.1155/2014/691480.
Full textAbstract:
The Bac-to-Bac baculovirus expression system can efficiently produce recombinant proteins, but the system may have to be optimized to achieve high-level expression for different candidate proteins. We reported here the efficient expression of acetylcholine-binding proteins from sea haresAplysia californica(Ac-AChBP) and a convenient method to monitor protein expression level in this expression system. Three key factors affecting expression of Ac-AChBP were optimized for maximizing the yield, which included the cell density, volume of the infecting baculovirus inoculums, and the culturing time of postinfection. We have found it to reach a high yield of ∼5 mg/L, which needs 55 h incubation after infection at the cell density of 2 × 106 cells/mL with an inoculum volume ratio of 1 : 100. The optimized expression system in this study was also applied for expressing another protein Ls-AChBP fromLymnaea stagnalissuccessfully. Therefore, this established method is helpful to produce high yields of AChBP proteins for X-ray crystallographic structural and functional studies.
3
Sandee, Duanpen, and Walter L. Miller. "High-Yield Expression of a Catalytically Active Membrane-Bound Protein: Human P450 Oxidoreductase." Endocrinology 152, no. 7 (2011): 2904–8. http://dx.doi.org/10.1210/en.2011-0230.
Full textAbstract:
P450 oxidoreductase (POR) is a two-flavin protein that reduces microsomal P450 enzymes and some other proteins. Preparation of active bacterially expressed human POR for biochemical studies has been difficult because membrane-bound proteins tend to interact with column matrices. To reduce column-protein interactions and permit more vigorous washing, human POR lacking 27 N-terminal residues (N-27 POR) was modified to carry a C-terminal Gly3His6-tag (N-27 POR-G3H6). When expressed in Escherichia coli, N-27 POR-G3H6 could be purified to apparent homogeneity by a modified, single-step nickel-nitrilotriacetic acid affinity chromatography, yielding 31 mg POR per liter of culture, whereas standard purification of native N-27 POR required multiple steps, yielding 5 mg POR per liter. Both POR proteins had absorption maxima at 375 and 453 nm and both reduced cytochrome c with indistinguishable specific activities. Using progesterone as substrate for bacterially expressed purified human P450c17, the Michaelis constant for 17α-hydroxylase activity supported by N-27 POR or N-27 POR-G3H6 were 1.73 or 1.49 μm, and the maximal velocity was 0.029 or 0.026 pmol steroids per picomole P450 per minute, respectively. Using 17-hydroxypregnenolone as the P450c17 substrate, the Michaelis constant for 17,20 lyase activity using N-27 POR or N-27 POR-G3H6 was 1.92 or 1.89 μm and the maximal velocity was 0.041 or 0.042 pmol steroid per picomole P450 per minute, respectively. Thus, N-27 POR-G3H6 is equally active as native N-27 POR. This expression and purification system permits the rapid preparation of large amounts of highly pure, biologically active POR and may be generally applicable for the preparation of membrane-bound proteins.
4
Feng, Ziru, Xifeng Li, Baofang Fan, Cheng Zhu, and Zhixiang Chen. "Maximizing the Production of Recombinant Proteins in Plants: From Transcription to Protein Stability." International Journal of Molecular Sciences 23, no. 21 (2022): 13516. http://dx.doi.org/10.3390/ijms232113516.
Full textAbstract:
The production of therapeutic and industrial recombinant proteins in plants has advantages over established bacterial and mammalian systems in terms of cost, scalability, growth conditions, and product safety. In order to compete with these conventional expression systems, however, plant expression platforms must have additional economic advantages by demonstrating a high protein production yield with consistent quality. Over the past decades, important progress has been made in developing strategies to increase the yield of recombinant proteins in plants by enhancing their expression and reducing their degradation. Unlike bacterial and animal systems, plant expression systems can utilize not only cell cultures but also whole plants for the production of recombinant proteins. The development of viral vectors and chloroplast transformation has opened new strategies to drastically increase the yield of recombinant proteins from plants. The identification of promoters for strong, constitutive, and inducible promoters or the tissue-specific expression of transgenes allows for the production of recombinant proteins at high levels and for special purposes. Advances in the understanding of RNAi have led to effective strategies for reducing gene silencing and increasing recombinant protein production. An increased understanding of protein translation, quality control, trafficking, and degradation has also helped with the development of approaches to enhance the synthesis and stability of recombinant proteins in plants. In this review, we discuss the progress in understanding the processes that control the synthesis and degradation of gene transcripts and proteins, which underlie a variety of developed strategies aimed at maximizing recombinant protein production in plants.
5
Qiao, Zilin, Yuejiao Liao, Mengyuan Pei, et al. "RSAD2 Is an Effective Target for High-Yield Vaccine Production in MDCK Cells." Viruses 14, no. 11 (2022): 2587. http://dx.doi.org/10.3390/v14112587.
Full textAbstract:
Increasingly, attention has focused on improving vaccine production in cells using gene editing technology to specifically modify key virus regulation-related genes to promote virus replication. In this study, we used DIA proteomics analysis technology to compare protein expression differences between two groups of MDCK cells: uninfected and influenza A virus (IAV) H1N1-infected cells 16 h post infection (MOI = 0.01). Initially, 266 differentially expressed proteins were detected after infection, 157 of which were upregulated and 109 were downregulated. We screened these proteins to 23 genes related to antiviral innate immunity regulation based on functional annotation database analysis and verified the mRNA expression of these genes using qPCR. Combining our results with published literature, we focused on the proteins RSAD2, KCNN4, IDO1, and ISG20; we verified their expression using western blot, which was consistent with our proteomics results. Finally, we knocked down RSAD2 using lentiviral shRNA expression vectors and found that RSAD2 inhibition significantly increased IAV NP gene expression, effectively promoting influenza virus replication with no significant effect on cell proliferation. These results indicate that RSAD2 is potentially an effective target for establishing high-yield vaccine MDCK cell lines and will help to fully understand the interaction mechanism between host cells and influenza viruses.
6
Kaipa, Jagan Mohan, Ganna Krasnoselska, Raymond J. Owens, and Joop van den Heuvel. "Screening of Membrane Protein Production by Comparison of Transient Expression in Insect and Mammalian Cells." Biomolecules 13, no. 5 (2023): 817. http://dx.doi.org/10.3390/biom13050817.
Full textAbstract:
Membrane proteins are difficult biomolecules to express and purify. In this paper, we compare the small-scale production of six selected eukaryotic integral membrane proteins in insect and mammalian cell expression systems using different techniques for gene delivery. The target proteins were C terminally fused to the green fluorescent marker protein GFP to enable sensitive monitoring. We show that the choice of expression systems makes a considerable difference to the yield and quality of the six selected membrane proteins. Virus-free transient gene expression (TGE) in insect High Five cells combined with solubilization in dodecylmaltoside plus cholesteryl hemisuccinate generated the most homogeneous samples for all six targets. Further, the affinity purification of the solubilized proteins using the Twin-Strep® tag improved protein quality in terms of yield and homogeneity compared to His-tag purification. TGE in High Five insect cells offers a fast and economically attractive alternative to the established methods that require either baculovirus construction and the infection of the insect cells or relatively expensive transient gene expression in mammalian cells for the production of integral membrane proteins.
7
Aslam, Muhammad Shahbaz, Iram Gull, Malik Siddique Mahmood та ін. "High yield expression, characterization, and biological activity of IFNα2-Tα1 fusion protein". Preparative Biochemistry & Biotechnology 50, № 3 (2019): 281–91. http://dx.doi.org/10.1080/10826068.2019.1689509.
Full text
8
Wang, Leyu, Meta Foster, Yan Zhang, et al. "High yield expression of non-phosphorylated protein tyrosine kinases in insect cells." Protein Expression and Purification 61, no. 2 (2008): 204–11. http://dx.doi.org/10.1016/j.pep.2008.05.017.
Full text
9
Sun, Jiakang, June A. Mayor, Rusudan Kotaria, and Ronald S. Kaplan. "High-Yield Expression and Purification of a Plasma Membrane Citrate Transport Protein." Biophysical Journal 96, no. 3 (2009): 686a. http://dx.doi.org/10.1016/j.bpj.2008.12.3624.
Full text
10
Chen, Xiaojing, Karyn L. Wilde, Hui Wang, et al. "High yield expression and efficient purification of deuterated human protein galectin-2." Food and Bioproducts Processing 90, no. 3 (2012): 563–72. http://dx.doi.org/10.1016/j.fbp.2011.12.004.
Full textDissertations / Theses on the topic "High-yield protein expression"
1
Zarate, Xristo, David Henderson, Keenan Phillips, April Lake, and David Galbraith. "Development of high-yield autofluorescent protein microarrays using hybrid cell-free expression with combined Escherichia coli S30 and wheat germ extracts." BioMed Central, 2010. http://hdl.handle.net/10150/610237.
Full textAbstract:
BACKGROUND:Protein-based microarray platforms offer considerable promise as high-throughput technologies in proteomics. Particular advantages are provided by self-assembling protein microarrays and much interest centers around analysis of eukaryotic proteins and their molecular interactions. Efficient cell-free protein synthesis is paramount for the production of self-assembling protein microarrays, requiring optimal transcription, translation, and protein folding. The Escherichia coli S30 extract demonstrates high translation rates but lacks the protein-folding efficiency of its eukaryotic counterparts derived from rabbit reticulocyte and wheat germ extract. In comparison to E. coli, eukaryotic extracts, on the other hand, exhibit slower translation rates and poor overall protein yields. A cell-free expression system that synthesizes folded eukaryotic proteins in considerable yields would optimize in vitro translation for protein microarray assembly.RESULTS:Self-assembling autofluorescent protein microarrays were produced by in situ transcription and translation of chimeric proteins containing a C-terminal Green Fluorescent Protein tag. Proteins were immobilized as array elements using an anti-GFP monoclonal antibody. The amounts of correctly-folded chimeric proteins were quantified by measuring the fluorescence intensity from each array element. During cell-free expression, very little or no fluorescence was observed from GFP-tagged multidomain eukaryotic plant proteins when in vitro translation was performed with E. coli S30 extract. Improvement was seen using wheat germ extract, but fluorescence intensities were still low because of poor protein yields. A hybrid in vitro translation system, combining S30 and wheat germ extracts, produced high levels of correctly-folded proteins for most of the constructs that were tested.CONCLUSION:The results are consistent with the hypothesis that the wheat germ extract enhances the protein folding capabilities of the in vitro system by providing eukaryotic ribosomes and chaperones and, at the same time, the E. coli S30 extract, which includes an ATP regeneration system, translates the polypeptides at high rates. This hybrid cell-free expression system allows the facile production of high-yield protein arrays suitable for downstream assays.
Book chapters on the topic "High-yield protein expression"
1
Graentzdoerffer, Andrea, Manfred Watzele, Bernd Buchberger, et al. "Optimization of the Translation Initiation Region of Prokaryotic Expression Vectors: High Yield In Vitro Protein Expression and mRNA Folding." In Cell-Free Translation Systems. Springer Berlin Heidelberg, 2002. http://dx.doi.org/10.1007/978-3-642-59379-6_20.
Full text
2
Palacio-Castañeda, Valentina, Roland Brock, and Wouter P. R. Verdurmen. "Generation of Protein-Phosphorodiamidate Morpholino Oligomer Conjugates for Efficient Cellular Delivery via Anthrax Protective Antigen." In Methods in Molecular Biology. Springer US, 2022. http://dx.doi.org/10.1007/978-1-0716-2010-6_8.
Full textAbstract:
AbstractPhosphorodiamidate morpholino oligomers (PMOs) offer great promise as therapeutic agents for translation blocking or splice modulation due to their high stability and affinity for target sequences. However, in spite of their neutral charge as compared to natural oligonucleotides or phosphorothioate analogs, they still show little permeability for cellular membranes, highlighting the need for effective cytosolic delivery strategies. In addition, the implementation of strategies for efficient cellular targeting is highly desirable to minimize side effects and maximize the drug dose at its site of action. Anthrax toxin is a three-protein toxin of which the pore-forming protein anthrax protective antigen (PA) can be redirected to a receptor of choice and lethal factor (LF), one of the two substrate proteins, can be coupled to various cargoes for efficient cytosolic cargo delivery. In this protocol, we describe the steps to produce the proteins and protein conjugates required for cytosolic delivery of PMOs through the cation-selective pore generated by anthrax protective antigen. The method relies on the introduction of a unique cysteine at the C-terminal end of a truncated LF (aa 1–254), high-yield expression of the (truncated) toxin proteins in E. coli, functionalization of a PMO with a maleimide group and coupling of the maleimide-functionalized PMO to the unique cysteine on LF by maleimide-thiol conjugation chemistry. Through co-administration of PA with LF-PMO conjugates, an efficient cytosolic delivery of PMOs can be obtained.
3
Xu, Jianfeng, and Marcia J. Kieliszewski. "A Novel Plant Cell Bioproduction Platform for High-Yield Secretion of Recombinant Proteins." In Recombinant Gene Expression. Humana Press, 2011. http://dx.doi.org/10.1007/978-1-61779-433-9_26.
Full text
4
Murray, Victoria, Yuefei Huang, Jianglei Chen, Jianjun Wang, and Qianqian Li. "A Novel Bacterial Expression Method with Optimized Parameters for Very High Yield Production of Triple-Labeled Proteins." In Methods in Molecular Biology. Humana Press, 2011. http://dx.doi.org/10.1007/978-1-61779-480-3_1.
Full text
5
Patel, Pryank, and Stuart Harbron. "Protein Expression and Production." In Molecular Biology and Biotechnology, 7th ed. The Royal Society of Chemistry, 2021. http://dx.doi.org/10.1039/9781788017862-00087.
Full textAbstract:
Knowledge of the full sequence of many genomes has led to the identification of thousands of genes encoding proteins with unknown or poorly known activity, which can only be elucidated by expression of the genes and analysis of the expressed protein by various methodologies. Producing recombinant proteins in forms that are either suitable for elucidating function for investigative purposes or in amounts useful for therapeutic applications is a key challenge. Approaches and hazards relating to the production of the protein in good yield and in the right form are evaluated, including consideration of host-related issues and the use of cell-free systems. Expression vectors, particularly pBAD and pET and their derivatives, are described, including their use in one-step cloning and expression systems. Fusion proteins formed from the protein of interest are appraised in relation to tags that enhance solubility and/or purification and the ease with which they may be subsequently removed. Consideration of eukaryotic and cell-free expression systems is also included. Finally, proteomic requirements through high-throughput methodologies are described.
6
Paterson, Andrew. "Application of the baculoviral expression system to signal transduction." In Signal Transduction. Oxford University PressOxford, 1999. http://dx.doi.org/10.1093/oso/9780199637218.003.0007.
Full textAbstract:
Abstract The Baculovirus Expression Vector System (BEYS) is an extremely versatile and powerful laboratory tool (1). In simplest terms, the BEYS allows the forced heterologous expression of recombinant protein in susceptible host strains of insect cells. Gene products from the simplest prokaryotes through to the higher eukaryotes have been expressed successfully in this system. Recombinant proteins are recovered in a folded and active form, and posttranslation modifications, such as phosphorylation, lipid acylation, and simple glycosylation are often preserved. Oligomerization is preserved also, as is the ability to form holomeric complexes. This has been demonstrated clearly with expression of the - 40 k Da G proteins in their native al3 ‘) ‘ heterotrimeric complexes (2), and extended to the activation of the kinase, c-Raf, upon coexpression with Ras and Src tyrosine kinase (3). Since the host insect cells maintain the ability to process eukaryotic pre-mRNA, heterologous expression from genomic DNA is possible. However, the most attractive facet of BEYS must be the high level of heterologous protein expression with values of between 0.1 and 30% of total cell protein being reported, and often with gene products that fail to express with activity or reasonable yield in other systems. The rationale of the BEYS is fairly simple. The baculoviral genome is engineered to incorporate the coding sequence of a foreign protein. Incorporation is directed to a position downstream, and under the control, of a strong viral promoter.
7
Thammacharoen, Sumpun, Nungnuch Saipin, Thiet Nguyen, and Narongsak Chaiyabutr. "Effects of High Ambient Temperature on Milk Protein Synthesis in Dairy Cows and Goats: Insights from the Molecular Mechanism Studies." In Milk Protein - New Research Approaches [Working Title]. IntechOpen, 2022. http://dx.doi.org/10.5772/intechopen.104563.
Full textAbstract:
Milk protein is well accepted for nutritional value compared with other sources of protein. Detailed understanding of the natural factors that can determine milk protein subcomponent (i.e., casein) not only fulfill the knowledge of protein synthesis but also provide the potential idea to improve milk quality. The variation in milk protein content from dairy cows and goats fed in tropical areas may determine the added value of milk from this region. Under prolonged high ambient temperature (HTa), dairy cows and goats are at the stage of heat stress. This physiological condition produces a negative effect on dairy cows and goats, i.e., food intake and milk yield. However, the higher milk protein content during summer is demonstrated in dairy goats in our condition. Likewise, an increase in heat shock protein 70 (Hsp70) gene expression from mammary epithelium cells isolated from either in vivo (summer and winter periods) and in vitro conditions suggests the direct effect of HTa on mammary gland and perhaps on milk protein synthesis. The intracellular effect of Hsp70 on milk protein synthesis has been proposed in regard to the endoplasmic reticulum and Golgi apparatus protein transportation and with the subcomponent of casein micelle. The present information reveals the molecular mechanism of HTa on milk protein synthesis.
8
Fonte, P., A. Malcangi, M. Stucchi, and A. Oppedisano. "Recombinant and Semisynthesis of Peptides." In Sustainability in Tides Chemistry. Royal Society of Chemistry, 2024. http://dx.doi.org/10.1039/9781837674541-00133.
Full textAbstract:
In the 1970s, recombinant deoxyribonucleic acid (rDNA) methods for cloning and expressing genes in microorganisms were developed and allowed the creation of various recombinant proteins with medicinal uses. The first therapeutically useful protein product resulting from recombinant DNA technology was human insulin obtained by Genentech. Its successful production in bacteria provided a practical, scalable source of human insulin and paved the way to the production of recombinant human hormones and their therapeutic use. rDNA technology and chemical synthesis are two possible manufacturing processes covering different areas of need. The rDNA approach is advantageous when peptides with longer sequences are produced, because yield and product purity do not depend on the peptide chain length. Biotechnology offers the possibility of large-scale peptide production at affordable cost using bacteria or yeasts as expression systems. There is no universal expression platform that is optimal for all therapeutic peptides and strong efforts have to be made to define it. An initial investment in research and development is mandatory to meet the target, but it is then rewarded by easy scale-up to the manufacturing plant. In terms of sustainability, biotechnology has a clear ecological advantage over classical industrial processes. Fermentation is a water-based process that uses waste from primary agriculture products as substrates and consumption of solvents in the purification step is relatively low. High cell density cultures are a preferred strategy to optimize recombinant protein volumetric productivity, which is a key parameter of bioprocess cost-effectiveness and sustainability.
9
Thapa, Bharti, and Abhisek Shrestha. "Protein Metabolism in Plants to Survive against Abiotic Stress." In Plant Defense Mechanisms [Working Title]. IntechOpen, 2022. http://dx.doi.org/10.5772/intechopen.102995.
Full textAbstract:
Plants are frequently subjected to several abiotic environmental stresses under natural conditions causing profound impacts on agricultural yield and quality. Plants can themselves develop a wide variety of efficient mechanisms to respond environmental challenges. Tolerance and acclimation of plants are always related to significant changes in protein, cellular localization, posttranscription, and posttranslational modifications. Protein response pathways as well as pathways unique to a given stress condition shared by plants under different stressed environment are discussed in this chapter. The various signaling of protein such as fluctuation, overexpression, and silencing of the protein gene are observed to be modulated in drought-tolerant plants. Similarly, gene expression, RNA processing, and metabolic process take place to cope with drought conditions. For adaption in water-submerged conditions, plants undergo reactive oxygen species (ROS), cell wall modification, proteolysis, and post-recovery protein metabolism. Heat shock protein and protein and lipid contents vary and play pivotal role in resisting low and high temperatures. In a nutshell, this paper provides an overview of several modification, synthesis, degradation, and metabolism of protein in plants to cope with and revive again to normal growing conditions against abiotic stress, emphasizing drought, submerged, extreme cold, and heat temperatures.
Conference papers on the topic "High-yield protein expression"
1
Lassner, Michael. "Ultra-High Protein Soybeans for Food and Aquaculture." In 2022 AOCS Annual Meeting & Expo. American Oil Chemists' Society (AOCS), 2022. http://dx.doi.org/10.21748/anya7548.
Full textAbstract:
Amfora is developing two categories of soybeans with increased protein content. One product is designed to increase the protein content of commodity soybeans by at least 10% with minimal impact on oil and yield. A second product is designed to deliver an ultra-high protein (ULTRAHIPRO TM) soymeal with more than 60% protein. A soybean meal with 60% protein can be used to displace soy protein concentrate in multiple applications including plant-based meats and as a high value ingredient in aquaculture feed. Amfora’s core technology is using gene editing to drive increased expression of NF-YC4, which results in a phenotype of increased protein at the expense of carbohydrates. We are using a combination of unique germplasm, modern genomics-based crop breeding and new breeding technologies to develop and commercialize valuable products for food and aquaculture applications.
2
Boyko, M. D., and G. V. Mkrtchyan. "Selection of cows according to the best milk protein genotypes in the highest breeding group using genetic evaluation methods." In Scientific achievements of the third millennium. НИЦ "LJournal", 2023. http://dx.doi.org/10.18411/satm-08-2023-06.
Full textAbstract:
Nowadays, the achievements of molecular genetics allow us to identify the genes that determine the expression of economically useful traits. Gene variant’s identifying will allow to conduct breeding work directly at the level of DNA in addition to the traditional selection. This article shows polymorphism and the frequency occurrence of alleles and genotypes by loci of 3 three milk protein genes has been identified, namely: κ-casein (CSN3), β-casein (CSN2) and β-lactoglobulin (LGB) in Holstein cattle. Based on the researching results it has been determined that most of high-productive animals have positive genotypes CSN3, CSN2 and LGB, which promotes the higher yield of quality production, accordingly, the level of economic efficiency increases.
3
Agouni, Abdelali, Duck Y. Lee, Assaad A. Eid, Yves Gorin, and Kumar Sharma. "The Protective Role of Sestrin2 in High Fat Diet-Induced Nephropathy." In Qatar University Annual Research Forum & Exhibition. Qatar University Press, 2020. http://dx.doi.org/10.29117/quarfe.2020.0134.
Full textAbstract:
Introduction: Obesity is a major risk factor for type-2 diabetes predisposing patients to diabetic nephropathy (DN), the leading cause of end-stage renal failure. Glomerular injury is a prominent pathological feature of DN. Sestrin2 (Sesn2) is a stress-induced protein, but its role in DN has not been investigated. Therefore, we have determined the impact of Sesn2 deletion in a mouse model of obesityinduced nephropathy. Materials and methods: We examined the effects of Sesn2-deficiency in a longterm (22 weeks) mouse model of high fat diet (HFD)-induced obesity on glomerular structure. The severity of renal injury and fibrosis in wild type (Sesn2+/+) mice (fed HFD or chow diets) was compared to that in Sesn2-deficient mice (Sesn2-/- ) fed HFD or chow diets. Animal work was carried out under an IACUC-approved protocol. Results: Data showed that Sesn2 ablation exacerbated HFD-induced glomerular fibrotic injury as evidenced by mesangial matrix hypertrophy and accumulation of both fibronectin and collagen IV. Western blot analysis revealed that HFD- or chow-fed Sesn2-/- mice exhibited higher protein expression of key lipogenic enzymes, fatty acid translocase CD36 (an indicator of lipid uptake), fatty acid synthase and ATP citrate lyase. Sesn2-deficiency in obese mice resulted in podocyte loss as indicated by reduced expression of synaptopodin. Glomerular lesions like those observed in HFD-fed wild-type mice were detected in Sesn2-/-mice fed a chow diet, indicating that the basal deletion of Sesn2 is deleterious by itself. Conclusions: We provide the first evidence that Sesn2 is renoprotective in obesity-induced nephropathy by diminishing lipid accumulation and blocking excessive lipid uptake and de novo lipid synthesis. Understanding the protective of Sesn2 should yield novel therapeutic interventions to effectively preserve glomerular function in obesity and diabetes.
4
Chang, Ting-Wei, Sheng-Hann Wang, Iuan Sheau Chin, et al. "Insect Odorant Binding Protein 2 Integrated with Flow Digital Nanoplasmon-metry for Neonicotinoid Pesticide Residues Sensing in Beverages." In Optical Manipulation and Its Applications. Optica Publishing Group, 2023. http://dx.doi.org/10.1364/oma.2023.am2d.4.
Full textAbstract:
Neonicotinoid is one class of the most used pesticides to repel pests. They demonstrated good efficiency but meanwhile pollute the ecosystem. Therefore, its abuse is an ever-thorny problem. Furthermore, since the multi-pesticide association is commonly used for high-efficient crop protection from pests in farms, quickly screening the pesticide contamination sorted by class is much more efficient and more accessible for on-site use. In this work, a novel and promising strategy that incorporated the neonicotinoid-specific odorant binding protein 2 (OBP2) with ultra-sensitive local surface plasmon resonance (LSPR)-based measurement, the flow digital nanoplasmon-metry (flow DiNM) was proposed. OBP2 modified on gold nanoparticles acted as the seizer to simultaneously capture neonicotinoids, such as imidacloprid, dinotefuran, and acetamiprid. The flow DiNM comprises spectral image contrast and digital analysis to enhance the slight LSPR change by the small molecule, neonicotinoid pesticides, attaching. It shows prominent LODs of 3.6, 7, and 15.3 ppb in imidacloprid, acetamiprid, and dinotefuran within the 45-minute detection. Furthermore, the blind tests show a high consistency to the standard method, and the recovery of true positives was 83% and 87.5% for green and black teas, respectively, and the recovery of true negatives can achieve 100 % with a total of 18 tests. Compared to conventional antibody-based immunoassay, the production of OBPS that uses E. coil protein expression takes advantage of high yield, time-saving, and cost-effectiveness. Together with its broad while specific neonicotinoid pesticides binding affinity and sensitivity of flow DiNM, this work demonstrated much higher accessibility to the on-site end users.
5
Irakleidis, Foivos, Michael Masucci, Eleftherios Sfakianakis, Peng H. Tan, and Hazel Welch. "MODIFIED SLOW DIGESTION TECHNIQUE FOR THE ISOLATION OF PATIENT-DERIVED CELLS: AN IN VITRO MODEL FOR THE DESIGN OF BREAST CANCER-ASSOCIATED STROMA." In Abstracts from the Brazilian Breast Cancer Symposium - BBCS 2021. Mastology, 2021. http://dx.doi.org/10.29289/259453942021v31s2018.
Full textAbstract:
Objective: Highly prevalent cancer-associated fibroblasts (CAFs) are understood to play a key role in tumorigenesis. Understanding of CAFs and tumor-associated stroma is considered to be essential in novel cancer therapies. Patient-derived cells (PDCs) more closely resemble tumor microenvironment compared with commercial cell lines that are subjected to genetic and phenotypic changes. However, PDCs use can be limited by challenges in isolating high-yield viable cultures. Overcoming these challenges would benefit novel personalized cancer research. In this study, we aimed to investigate the effectiveness of modified tissue digestion processing techniques of isolation of PDCs. Methodology: PDCs were isolated from breast tissues collected from patients who had previously been diagnosed with breast cancer. Modification of slow and fast digestion processing techniques was used, followed by analysis for morphology and protein marker expression. Results: Isolated PDCs were presented with different morphologies and functions compared with breast cancer cell lines. Higher growth potential was observed with a combination of maintenance and filtered conditioned medium. High expression of Vimentin and morphological characteristics of spindle-shaped large cells confirmed the PDCs as fibroblasts. The modified slow digestion approach used in this study was successful in isolating fibroblasts from retrieved breast tissue. The fast digestion approach was not viable and was abandoned early due to poor production of cells. Conclusions: PDCs were isolated using a modified slow digestion approach. PDC cultures can more effectively represent breast cancer stroma and are becoming an essential platform for research as a personalized in vitro model for molecular breast cancer research. This study presents a highly successful method of isolating PDCs from breast cancer patients.
6
Utkina, A. A., A. A. Kudryavtseva, and I. V. Manuhkov. "HIGH-YIELD PRODUCTION OF “DIFFICULT-TO-EXPRESS” PROTEINS ARDA AND ARDB WITH THE THERMO-INDUCIBLE VECTOR PIR-DPAL." In X Международная конференция молодых ученых: биоинформатиков, биотехнологов, биофизиков, вирусологов и молекулярных биологов — 2023. Novosibirsk State University, 2023. http://dx.doi.org/10.25205/978-5-4437-1526-1-379.
Full textAbstract:
The antirestriction proteins ArdA and ArdB inhibit type I restriction-modification (RM) systems. Such proteins are usually difficult to obtain in Escherichia coli cells in large quantities since they are toxic to the host cell. This work is devoted to the cloning of the ardA and ardB antirestrictase genes into the thermo-inducible vector pIR-DPAl vector, which made it possible to increase the efficiency of its expression and purification.
Reports on the topic "High-yield protein expression"
1
Chanprateep Napathorn, Suchada, and Tanapat Palaga. Expression of novel fusion proteins IL2/FU-MK-1-scFv in microorganism-host cells and its potential anti-tumor activities as a cytotoxic immunotherapy agent for FU-MK-1 expressing tumors. Chulalongkorn University, 2012. https://doi.org/10.58837/chula.res.2012.25.
Full textAbstract:
MK-1, the target molecule of FU-MK-1, is encoded by the GA733-2 gene, which is currently being used as a target in clinical trials for gastric, intestinal, and biliary cancer treatment with monoclonal antibodies. Here, two different arrangement of heavy-chain and K light-chain variable fusion gene, IL2/FUscFv(VK-VH) or IL2/FUscFv(VWVK), were constructed. The efficiency of protein expression in prokaryotic host expression system, Escherichia coli strains BL21(DE3)pLysS and Rosetta-gami B was compared with eukaryotic host expression system, Pichia pastoris strains GS115 and KM71H, for their ability to produce fusion protein. It was found that the fusion proteins were expressed in E. coli strain Rosetta-gami B in reasonable yield with high percentage of soluble form (0.25 gil of IL2/FUscFv(Vw VK) with solubility 89.29% and 0.26 gil of IL2/FUscFv(VK-VH) with solubility 84.61 %) when cultivated at 25°C, pH 7, and induced with 0.05 mM IPTG for 10 h. In comparison, P. postoris produced the secreted fusion proteins. The highest production of the fusion protein at 0.258 ± 0.013 gil was obtained under pH 3, 30°C, and induction with 0.1 % (v/v) methanol for 96 h in shaken flask cultivation. Finally, fed-batch cultivation was performed in 5 l fermenter. The highest amount of secreted fusion protein was 0.425 ± 0.002 gil. Following purification, the fusion protein was examined the specific binding activity to CHO cell expressing MK-1. The fusion protein retained the specific binding activity to MK-1 antigen due to it significantly bound to MK-1 expressing CHO cell but not MK-1 non-expressing CHO cell when compared using Student's t test (p<0 .05).
2
Blum, Abraham, Henry T. Nguyen, and N. Y. Klueva. The Genetics of Heat Shock Proteins in Wheat in Relation to Heat Tolerance and Yield. United States Department of Agriculture, 1993. http://dx.doi.org/10.32747/1993.7568105.bard.
Full textAbstract:
Fifty six diverse spring wheat cultivars were evaluated for genetic variation and heritability for thermotolerance in terms of cell-membrane stability (CMS) and triphenyl tetrazolium chloride (TTC) reduction. The most divergent cultivars for thermotolerance (Danbata-tolerant and Nacozari-susceptible) were crossed to develop an F8 random onbred line (RIL) population. This population was evaluated for co-segragation in CMS, yield under heat stress and HSP accumulation. Further studies of thermotolerance in relations to HSP and the expression of heterosis for growth under heat stress were performed with F1 hybrids of wheat and their parental cultivars. CMS in 95 RILs ranged from 76.5% to 22.4% with 71.5% and 31.3% in Danbata and Nacozari, respectively. The population segregated with a normal distribution across the full range of the parental values. Yield and biomass under non-stress conditions during the normal winter season at Bet Dagan dit not differ between the two parental cultivar, but the range of segregation for these traits in 138 RILs was very high and distinctly transgressive with a CV of 35.3% and 42.4% among lines for biomass and yield, respectively. Mean biomass and yield of the population was reduced about twofold when grown under the hot summer conditions (irrigated) at Bet Dagan. Segregation for biomass and yield was decreased relative to the normal winter conditions with CV of 20.2% and 23.3% among lines for biomass and yield, respectively. However, contrary to non-stress conditions, the parental cultivars differed about twofold in biomass and yield under heat stress and the population segregated with normal distribution across the full range of this difference. CMS was highly and positively correlated across 79 RILs with biomass (r=0.62**) and yield (r=0.58**) under heat stress. No such correlation was obtained under the normal winter conditions. All RILs expressed a set of HSPs under heat shock (37oC for 2 h). No variation was detected among RILs in high molecular weight HSP isoforms and they were similar to the patterns of the parental cultivars. There was a surprisingly low variability in low molecular weight HSP isoforms. Only one low molecular weight and Nacozari-specific HSP isoform (belonging to HSP 16.9 family) appeared to segregate among all RILs, but it was not quantitatively correlated with any parameter of plant production under heat stress or with CMS in this population. It is concluded that this Danbata/Nacozari F8 RIL population co-segregated well for thermotolerance and yield under heat stress and that CMS could predict the relative productivity of lines under chronic heat stress. Regretfully this population did not express meaningful variability for HSP accumulation under heat shock and therefore no role could be seen for HSP in the heat tolerance of this population. In the study of seven F1 hybrids and their parent cultivars it was found that heterosis (superiority of the F1 over the best parent) for CMs was generally lower than that for growth under heat stress. Hybrids varied in the rate of heterosis for growth at normal (15o/25o) and at high (25o/35o) temperatures. In certain hybrids heterosis for growth significantly increased at high temperature as compared with normal temperature, suggesting temperature-dependent heterosis. Generally, under normal temperature, only limited qualitative variation was detected in the patterns of protein synthesis in four wheat hybrids and their parents. However, a singular protein (C47/5.88) was specifically expressed only in the most heterotic hybrid at normal temperature but not in its parent cultivars. Parental cultivars were significantly different in the sets of synthesized HSP at 37o. No qualitative changes in the patterns of protein expression under heat stress were correlated with heterosis. However, a quantitative increase in certain low molecular weight HSP (mainly H14/5.5 and H14.5.6, belonging to the HSP16.9 family) was positively associated with greater heterosis for growth at high temperature. None of these proteins were correlated with CMS across hybrids. These results support the concept of temperature-dependent heterosis for growth and a possible role for HSP 16.9 family in this respect. Finally, when all experiments are viewed together, it is encouraging to find that genetic variation in wheat yield under chronic heat stress is associated with and well predicted by CMS as an assay of thermotolerance. On the other hand the results for HSP are elusive. While very low genetic variation was expressed for HSP in the RIL population, a unique low molecular weight HSP (of the HSP 16.9 family) could be associated with temperature dependant heterosis for growth.
3
Savaldi-Goldstein, Sigal, and Todd C. Mockler. Precise Mapping of Growth Hormone Effects by Cell-Specific Gene Activation Response. United States Department of Agriculture, 2012. http://dx.doi.org/10.32747/2012.7699849.bard.
Full textAbstract:
Plant yield largely depends on a complex interplay and feedback mechanisms of distinct hormonal pathways. Over the past decade great progress has been made in elucidating the global molecular mechanisms by which each hormone is produced and perceived. However, our knowledge of how interactions between hormonal pathways are spatially and temporally regulated remains rudimentary. For example, we have demonstrated that although the BR receptor BRI1 is widely expressed, the perception of BRs in epidermal cells is sufficient to control whole-organ growth. Supported by additional recent works, it is apparent that hormones are acting in selected cells of the plant body to regulate organ growth, and furthermore, that local cell-cell communication is an important mechanism. In this proposal our goals were to identify the global profile of translated genes in response to BR stimulation and depletion in specific tissues in Arabidopsis; determine the spatio-temporal dependency of BR response on auxin transport and signaling and construct an interactive public website that will provide an integrated analysis of the data set. Our technology incorporated cell-specific polysome isolation and sequencing using the Solexa technology. In the first aim, we generated and confirmed the specificity of novel transgenic lines expressing tagged ribosomal protein in various cell types in the Arabidopsis primary root. We next crossed these lines to lines with targeted expression of BRI1 in the bri1 background. All lines were treated with BRs for two time points. The RNA-seq of their corresponding immunopurified polysomal RNA is nearly completed and the bioinformatic analysis of the data set will be completed this year. Followed, we will construct an interactive public website (our third aim). In the second aim we started revealing how spatio-temporalBR activity impinges on auxin transport in the Arabidopsis primary root. We discovered the unexpected role of BRs in controlling the expression of specific auxin efflux carriers, post-transcriptionally (Hacham et al, 2012). We also showed that this regulation depends on the specific expression of BRI1 in the epidermis. This complex and long term effect of BRs on auxin transport led us to focus on high resolution analysis of the BR signaling per se. Taking together, our ongoing collaboration and synergistic expertise (hormone action and plant development (IL) and whole-genome scale data analysis (US)) enabled the establishment of a powerful system that will tell us how distinct cell types respond to local and systemic BR signal. BR research is of special agriculture importance since BR application and BR genetic modification have been shown to significantly increase crop yield and to play an important role in plant thermotolerance. Hence, our integrated dataset is valuable for improving crop traits without unwanted impairment of unrelated pathways, for example, establishing semi-dwarf stature to allow increased yield in high planting density, inducing erect leaves for better light capture and consequent biomass increase and plant resistance to abiotic stresses.
4
Guy, Charles, Gozal Ben-Hayyim, Gloria Moore, Doron Holland, and Yuval Eshdat. Common Mechanisms of Response to the Stresses of High Salinity and Low Temperature and Genetic Mapping of Stress Tolerance Loci in Citrus. United States Department of Agriculture, 1995. http://dx.doi.org/10.32747/1995.7613013.bard.
Full textAbstract:
The objectives that were outlined in our original proposal have largely been achieved or will be so by the end of the project in February 1995 with one exception; that of mapping cold tolerance loci based on the segregation of tolerance in the BC1 progeny population. Briefly, our goals were to 1) construct a densely populated linkage map of the citrus genome: 2) map loci important in cold and/or salt stress tolerance; and 3) characterize the expression of genes responsive to cold land salt stress. As can be seen by the preceding listing of accomplishments, our original objectives A and B have been realized, objective C has been partially tested, objective D has been completed, and work on objectives E and F will be completed by the end of 1995. Although we have yet to map any loci that contribute to an ability of citrus to maintain growth when irrigated with saline water, our very encouraging results from the 1993 experiment provides us with considerable hope that 1994's much more comprehensive and better controlled experiment will yield the desired results once the data has been fully analyzed. Part of our optimism derives from the findings that loci for growth are closely linked with loci associated with foliar Cl- and Na+ accumulation patterns under non-salinization conditions. In the 1994 experiment, if ion exclusion or sequestration traits are segregating in the population, the experimental design will permit their resolution. Our fortunes with respect to cold tolerance is another situation. In three attempts to quantitatively characterize cold tolerance as an LT50, the results have been too variable and the incremental differences between sensitive and tolerant too small to use for mapping. To adequately determine the LT50 requires many plants, many more than we have been able to generate in the time and space available by making cuttings from small greenhouse-grown stock plants. As it has turned out, with citrus, to prepare enough plants needed to be successful in this objective would have required extensive facilities for both growing and testing hardiness which simply were not available at University of Florida. The large populations necessary to overcome the variability we encountered was unanticipated and unforeseeable at the project's outset. In spite of the setbacks, this project, when it is finally complete will be exceedingly successful. Listing of Accomplishments During the funded interval we have accomplished the following objectives: Developed a reasonably high density linkage map for citrus - mapped the loci for two cold responsive genes that were cloned from Poncirus - mapped the loci for csa, the salt responsive gene for glutathione peroxidase, and ccr a circadian rhythm gene from citrus - identified loci that confer parental derived specific DNA methylation patterns in the Citrus X Poncirus cross - mapped 5 loci that determine shoot vigor - mapped 2 loci that influence leaf Na+ accumulation patterns under non-saline conditions in the BC1 population - mapped 3 loci that influence leaf Na+ accumulation paterns during salt sress - mapped 2 loci that control leaf Cl- accumulation patterns under non-saline conditions - mapped a locus that controls leaf Cl- accumulation patterns during salt stress Screened the BC1 population for growth reduction during salinization (controls and salinized), and cold tolerance - determined population variation for shoot/root ratio of Na+ and Cl- - determined levels for 12 inorganic nutrient elements in an effort to examine the influence of salinization on ion content with emphasis on foliar responses - collected data on ion distribution to reveal patterns of exclusion/sequestration/ accumulation - analyzed relationships between ion content and growth Characterization of gene expression in response to salt or cold stress - cloned the gene for the salt responsive protein csa, identified it as glutathione peroxidase, determined the potential target substrate from enzymatic studies - cloned two other genes responsive to salt stress, one for the citrus homologue of a Lea5, and the other for an "oleosin" like gene - cold regulated (cor) genes belonging to five hybridization classes were isolated from Poncirus, two belonged to the group 2 Lea superfamily of stress proteins, the others show no significant homology to other known sequences - the expression of csa during cold acclimation was examined, and the expression of some of the cor genes were examined in response to salt stress - the influence of salinization on cold tolerance has been examined with seedling populations - conducted protein blot studies for expression of cold stress proteins during salt stress and vice versa
5
Yaron, Zvi, Martin P. Schreibman, Abigail Elizur, and Yonathan Zohar. Advancing Puberty in the Black Carp (Mylopharyngodon Piceus) and the Striped Bass (Morone Saxatilis). United States Department of Agriculture, 1993. http://dx.doi.org/10.32747/1993.7568102.bard.
Full textAbstract:
The black carp (bc)GtH IIb cDNA was amplified and isolated, cloned and sequenced. Comparison of the bcGtH IIb deduced a.a. sequence with that of GtH IIb from other teleosts revealed high homology to cyprinid species and a lower homology to salmonid or perciform fish. The gene coding for the GtH IIb was isolated and sequenced. Three bc recombinant phages which hybridized to the goldfish GtH Ib cDNA probe were isolated and are currently being characterized. The region coding for the mature GtH IIb was expressed in a bacterial expression vector resulting in the production of a recombinant protein. In vitro folding resulted in a protein only 1.3% of which displaced the native common carp GtH II in a RIA. Therefore, the common carp GtH RIA was utilized for the physiological studies at the current phase of the project. Two non-functional sites were identified along the brain-pituitary gonadal axis in the immature black carp. The pituitary is refractory to GnRH stimulation due to a block proximal to the activation of PKA and PKC probably at the level of GnRH receptors. The gonads, although capable of producing steroids, are refractory to gonadotropic stimulation but do respond to cAMP antagonists, indicating a block at the GtH receptor level. Attempts to advance puberty in 2 and 3 y old black carp showed that testosterone (T) stimulates GtH synthesis in the pituitary and increases its sensitivity to GnRh. A 2 month treatment combining T+GnRH increased the circulating GFtH level in 3 y old fish. Addition of domperidone to such a treatment facilitated both the accumulation of GtH in the pituitary and its response to GnRH. The cDNA of striped bass GtH a, Ib and IIb subunits were amplified, isolated, cloned and sequenced, and their deduced a.a. sequences were compared with those of other teleosts. A ribonuclease protection assay was developed for a sensitive and simultaneous determination of all GtH subunits, and of b-actin mRNAs of the striped bass. GnRH stimulated dramatically the expression of the a and GtH IIb subunits but the level of GtH Ib mRNA increased only moderately. These findings suggest that GtH-II, considered in salmonids to be involved only in final stages of gametogenesis, can be induced by GnRH to a higher extent than GtH-I in juvenile striped bass. The native GtH II of the striped bass was isolated and purified, and an ELISA for its determination was developed. The production of all recombinant striped bass GtH subunits is in progress using the insect cell (Sf9) culture and the BAC-TO-BAC baculovirus expression system. A recombinant GtH IIb subunit has been produced already, and its similarity to the native subunit was confirmed. The yield of the recombinant glycoprotein can reach 3.5 mg/ml after 3 days culture. All male striped bass reach puberty after 3 y. However, precocious puberty was discovered in 1 and 2 y old males. Females become vitellogenic during their 4th year. In immature 2 y old females, T treatment elevates the pituitary GtH II content while GnRH only potentiates the effect. However, in males GnRH and not T affects GtH accumulation in the pituitary. Neither GnRH, nor T treatment resulted in gonadal growth in 2 y old striped bass, indicating that either the accumulated GtH II was not released, or if released, the gonads were refractory to GtH stimulation, similar to the situation in the immature black carp. In 3 y old female striped bass, 150 day GnRHa treatment resulted in an increase in GSI, while T treatment, with or without GnRHa, resulted in a decrease in oocyte diameter, similar to the effect seen in the black carp. Further attempts to advance puberty in both fish species should take into account the positive effect of T on pituitary GtH and its negative effect of ovarian growth.
6
Halevy, Orna, Sandra Velleman, and Shlomo Yahav. Early post-hatch thermal stress effects on broiler muscle development and performance. United States Department of Agriculture, 2013. http://dx.doi.org/10.32747/2013.7597933.bard.
Full textAbstract:
In broilers, the immediate post-hatch handling period exposes chicks to cold or hot thermal stress, with potentially harmful consequences to product quantity and quality that could threaten poultry meat marketability as a healthy, low-fat food. This lower performance includes adverse effects on muscle growth and damage to muscle structure (e.g., less protein and more fat deposition). A leading candidate for mediating the effects of thermal stress on muscle growth and development is a unique group of skeletal muscle cells known as adult myoblasts (satellite cells). Satellite cells are multipotential stem cells that can be stimulated to follow other developmental pathways, especially adipogenesis in lieu of muscle formation. They are most active during the first week of age in broilers and have been shown to be sensitive to environmental conditions and nutritional status. The hypothesis of the present study was that immediate post-hatch thermal stress would harm broiler growth and performance. In particular, growth characteristics and gene expression of muscle progenitor cells (i.e., satellite cells) will be affected, leading to increased fat deposition, resulting in long-term changes in muscle structure and a reduction in meat yield. The in vitro studies on cultured satellite cells derived from different muscle, have demonstrated that, anaerobic pectoralis major satellite cells are more predisposed to adipogenic conversion and more sensitive during myogenic proliferation and differentiation than aerobic biceps femoris cells when challenged to both hot and cold thermal stress. These results corroborated the in vivo studies, establishing that chronic heat exposure of broiler chicks at their first two week of life leads to impaired myogenicity of the satellite cells, and increased fat deposition in the muscle. Moreover, chronic exposure of chicks to inaccurate temperature, in particular to heat vs. cold, during their early posthatch periods has long-term effects of BW, absolute muscle growth and muscle morphology and meat quality. The latter is manifested by higher lipid and collagen deposition and may lead to the white striping occurrence. The results of this study emphasize the high sensitivity of muscle progenitor cells in the early posthatch period at a time when they are highly active and therefore the importance of rearing broiler chicks under accurate ambient temperatures. From an agricultural point of view, this research clearly demonstrates the immediate and long-term adverse effects on broiler muscling and fat formation due to chronic exposure to hot stress vs. cold temperatures at early age posthatch. These findings will aid in developing management strategies to improve broiler performance in Israel and the USA. BARD Report - Project4592 Page 2 of 29
7
Weller, Joel I., Derek M. Bickhart, Micha Ron, Eyal Seroussi, George Liu, and George R. Wiggans. Determination of actual polymorphisms responsible for economic trait variation in dairy cattle. United States Department of Agriculture, 2015. http://dx.doi.org/10.32747/2015.7600017.bard.
Full textAbstract:
The project’s general objectives were to determine specific polymorphisms at the DNA level responsible for observed quantitative trait loci (QTLs) and to estimate their effects, frequencies, and selection potential in the Holstein dairy cattle breed. The specific objectives were to (1) localize the causative polymorphisms to small chromosomal segments based on analysis of 52 U.S. Holstein bulls each with at least 100 sons with high-reliability genetic evaluations using the a posteriori granddaughter design; (2) sequence the complete genomes of at least 40 of those bulls to 20 coverage; (3) determine causative polymorphisms based on concordance between the bulls’ genotypes for specific polymorphisms and their status for a QTL; (4) validate putative quantitative trait variants by genotyping a sample of Israeli Holstein cows; and (5) perform gene expression analysis using statistical methodologies, including determination of signatures of selection, based on somatic cells of cows that are homozygous for contrasting quantitative trait variants; and (6) analyze genes with putative quantitative trait variants using data mining techniques. Current methods for genomic evaluation are based on population-wide linkage disequilibrium between markers and actual alleles that affect traits of interest. Those methods have approximately doubled the rate of genetic gain for most traits in the U.S. Holstein population. With determination of causative polymorphisms, increasing the accuracy of genomic evaluations should be possible by including those genotypes as fixed effects in the analysis models. Determination of causative polymorphisms should also yield useful information on gene function and genetic architecture of complex traits. Concordance between QTL genotype as determined by the a posteriori granddaughter design and marker genotype was determined for 30 trait-by-chromosomal segment effects that are segregating in the U.S. Holstein population; a probability of <10²⁰ was used to accept the null hypothesis that no segregating gene within the chromosomal segment was affecting the trait. Genotypes for 83 grandsires and 17,217 sons were determined by either complete sequence or imputation for 3,148,506 polymorphisms across the entire genome. Variant sites were identified from previous studies (such as the 1000 Bull Genomes Project) and from DNA sequencing of bulls unique to this project, which is one of the largest marker variant surveys conducted for the Holstein breed of cattle. Effects for stature on chromosome 11, daughter pregnancy rate on chromosome 18, and protein percentage on chromosome 20 met 3 criteria: (1) complete or nearly complete concordance, (2) nominal significance of the polymorphism effect after correction for all other polymorphisms, and (3) marker coefficient of determination >40% of total multiple-regression coefficient of determination for the 30 polymorphisms with highest concordance. The missense polymorphism Phe279Tyr in GHR at 31,909,478 base pairs on chromosome 20 was confirmed as the causative mutation for fat and protein concentration. For effect on fat percentage, 12 additional missensepolymorphisms on chromosome 14 were found that had nearly complete concordance with the suggested causative polymorphism (missense mutation Ala232Glu in DGAT1). The markers used in routine U.S. genomic evaluations were increased from 60,000 to 80,000 by adding markers for known QTLs and markers detected in BARD and other research projects. Objectives 1 and 2 were completely accomplished, and objective 3 was partially accomplished. Because no new clear-cut causative polymorphisms were discovered, objectives 4 through 6 were not completed.
8
Cohen, Roni, Kevin Crosby, Menahem Edelstein, et al. Grafting as a strategy for disease and stress management in muskmelon production. United States Department of Agriculture, 2004. http://dx.doi.org/10.32747/2004.7613874.bard.
Full textAbstract:
The overall objective of this research was to elucidate the horticultural, pathological, physiological and molecular factors impacting melon varieties (scion) grafted onto M. cannonballus resistant melon and squash rootstocks. Specific objectives were- to compare the performance of resistant melon germplasm (grafted and non-grafted) when exposed to M. cannoballus in the Lower Rio Grande valley and the Wintergarden, Texas, and in the Arava valley, Israel; to address inter-species relationships between a Monosporascus resistant melon rootstock and susceptible melon scions in terms of fruit-set, fruit quality and yield; to study the factors which determine the compatibility between the rootstock and the scion in melon; to compare the responses of graft unions of differing compatibilities under disease stress, high temperatures, deficit irrigation, and salinity stress; and to investigate the effect of rootstock on stress related gene expression in the scion. Some revisions were- to include watermelon in the Texas investigations since it is much more economically important to the state, and also to evaluate additional vine decline pathogens Didymella bryoniae and Macrophomina phaseolina. Current strategies for managing vine decline rely heavily on soil fumigation with methyl bromide, but restrictions on its use have increased the need for alternative management strategies. Grafting of commercial melon varieties onto resistant rootstocks with vigorous root systems is an alternative to methyl bromide for Monosporascus root rot/vine decline (MRR/VD) management in melon production. Extensive selection and breeding has already produced potential melon rootstock lines with vigorous root systems and disease resistance. Melons can also be grafted onto Cucurbita spp., providing nonspecific but efficient protection from a wide range of soil-borne diseases and against some abiotic stresses, but compatibility between the scion and the rootstock can be problematic. During the first year experiments to evaluate resistance to the vine decline pathogens Monosporascus cannonballus, Didymella bryoniae, and Macrophomina phaseolina in melon and squash rootstocks proved the efficacy of these grafted plants in improving yield and quality. Sugars and fruit size were better in grafted versus non-grafted plants in both Texas and Israel. Two melons (1207 and 124104) and one pumpkin, Tetsukabuto, were identified as the best candidate rootstocks in Texas field trials, while in Israel, the pumpkin rootstock RS59 performed best. Additionally, three hybrid melon rootstocks demonstrated excellent resistance to both M. cannonballus and D. bryoniae in inoculated tests, suggesting that further screening for fruit quality and yield should be conducted. Experiments with ABA in Uvalde demonstrated a significant increase in drought stress tolerance and concurrent reduction in transplant shock due to reduced transpiration for ‘Caravelle’ plants. In Israel, auxin was implicated in reducing root development and contributing to increased hydrogen peroxide, which may explain incompatibility reactions with some squash rootstocks. However, trellised plants responded favorably to auxin (NAA) application at the time of fruit development. Gene expression analyses in Israel identified several cDNAs which may code for phloem related proteins, cyclins or other factors which impact the graft compatibility. Manipulation of these genes by transformation or traditional breeding may lead to improved rootstock cultivars. Commercial applications of the new melon rootstocks as well as the ABA and TIBA growth regulators have potential to improve the success of grafted melons in both Israel and Texas. The disease resistance, fruit quality and yield data generated by the field trials will help producers in both locations to decide what rootstock/scion combinations will be best.