Articoli di riviste sul tema "Promotor <Genetik>"
Segui questo link per vedere altri tipi di pubblicazioni sul tema: Promotor <Genetik>.
Cita una fonte nei formati APA, MLA, Chicago, Harvard e in molti altri stili
Vedi i top-50 articoli di riviste per l'attività di ricerca sul tema "Promotor <Genetik>".
Accanto a ogni fonte nell'elenco di riferimenti c'è un pulsante "Aggiungi alla bibliografia". Premilo e genereremo automaticamente la citazione bibliografica dell'opera scelta nello stile citazionale di cui hai bisogno: APA, MLA, Harvard, Chicago, Vancouver ecc.
Puoi anche scaricare il testo completo della pubblicazione scientifica nel formato .pdf e leggere online l'abstract (il sommario) dell'opera se è presente nei metadati.
Vedi gli articoli di riviste di molte aree scientifiche e compila una bibliografia corretta.
1
Oktariana, Desi, Irsan Saleh, Zen Hafy e Iche Andriyani Liberty. "Peran Polimorfisme Promotor Gen Interleukin-10 pada Penyakit Kusta: Tinjauan Sistematik". Journal Of The Indonesian Medical Association 73, n. 1 (5 maggio 2023): 15–24. http://dx.doi.org/10.47830/jinma-vol.73.1-2023-816.
Testo completoAbstract (sommario):
Pendahuluan: Kusta masih merupakan masalah penting di Indonesia karena jumlah kasus yang terus bertambah dan belum tereliminasi hingga saat ini. Salah satu sitokin yang berperan dalam kusta adalah interleukin-10 (IL-10), yang memiliki efek dominan anti-inflamasi dan mempengaruhi imunopatogenesis kusta. Faktor genetik, termasuk polimorfisme promotor gen IL-10, mempengaruhi jumlah IL-10 yang dihasilkan dan juga berkontribusi pada risiko terjadinya kusta. Oleh karena itu, penelitian ini bertujuan untuk memperoleh informasi tentang peran polimorfisme promotor gen IL-10 dalam kusta.Metode: Dalam penulisan ini, digunakan metode tinjauan sistematik yang bertujuan untuk mengidentifikasi, mengevaluasi, dan membahas secara mendalam karyakarya yang telah dipublikasikan melalui jurnal nasional dan internasional seperti Google Scholar, Science Direct, Elsevier, EBSCO, Medline, PubMed, Proquest, dan Wiley. Penelitian ini dilakukan dengan melakukan pencarian terhadap database tersebut menggunakan kata kunci yang relevan dengan topik penelitian. Setelah itu, dilakukan seleksi artikel berdasarkan kriteria inklusi dan eksklusi yang telah ditetapkan. Artikel yang memenuhi kriteria tersebut kemudian dievaluasi secara kualitas untuk memastikan keakuratan dan keandalan informasi yang disajikan. Akhirnya, hasil tinjauan sistematik ini digunakan untuk memperoleh informasi yang akurat dan terkini tentang peran polimorfisme promotor gen IL-10 dalam kusta.Hasil: Sebanyak 14 artikel dikaji dalam tinjauan sistematik ini dan didapatkan hasil yang beragam pada setiap populasi penelitian. Namun, terdapat kecenderungan bahwa polimorfisme promotor gen IL-10 terkait dengan penyakit kusta, yang ditemukan pada sebagian besar populasi penelitian.Kesimpulan: Polimorfisme promotor gen IL-10 cenderung terkait dengan penyakit kusta.
2
Wurlina, Wurlina, Rimayanti Rimayanti, Mas'ud Hariadi e Dewa Ketut Meles. "PEMBIBITAN DAN PENGGEMUKAN KAMBING “LOKETAWA” PENGHASIL DAGING DAN SUSU RAKITAN TEKNOBREEDING DAN TEKNOFATTENING PAKAN TANPA HIJAUAN (COMPLETE FEED)". Jurnal Layanan Masyarakat (Journal of Public Services) 1, n. 1 (1 giugno 2017): 46. http://dx.doi.org/10.20473/jlm.v1i1.2017.46-50.
Testo completoAbstract (sommario):
IbM to business gorup in the goat breeding and fattening “Loketawa” aims to: 1) improve local goat genetics through IB using Etawa stud 2) obtain mother goats lust together done synchronization lusts 3) get mother goat bunting from once a year to 2 times a year 4) increase the number of children from 1 birth to 3-4 births using superovulation techniques 5) reduce production costs, make feed without forage and growth promotor. The method of implementation used 1) introducing stud Etawa meat and milk producers 2) synchronization lust using PGF2α 3) superovulation using hormone FSH and LH 4) IB on goats using Etawa goat cuttings, 5) the processing of feed without forage and growth promoter. The results are as follows: 1) as many as 20 heads of goats are simultaneously using 100% PGF2α, artificial insemination on 10 heads of goats without super ovulation has an average child of 1.6 births, while artificial insemination on 10 goats with super ovulation having an average child 3 births, 3) an average goat weight increase of 252.35 grams per day. AbstrakIpteks bagi Masyarakat yang dilakukan pada kelompok usaha pembibitan dan penggemukan kambing “Loketawa” bertujuan: 1) memperbaiki genetik kambing lokal melalui IB menggunakan pejantan Etawa 2) mendapatkan induk kambing birahi bersamaam dilakukan sinkronisasi birahi 3) mendapatkan induk kambing bunting dari setahun sekali menjadi 2 kali setahun 4) meningkatkan jumlah anak dari 1 ekor sekelahiran menjadi 3-4 ekor sekelahiran menggunakan teknik superovulasi 5) menekan biaya produksi, membuat pakan tanpa hijauan dan growth promotor. Metode pelaksanaan yang digunakan adalah 1) memperkenalkan pejantan Etawa penghasil daging dan susu 2) sinkronisasi birahi menggunakan PGF2α 3) superovulasi menggunakan hormon FSH dan LH 4) IB pada kambing menggunakan semen kambing Etawa, 5) pengolahan pakan tanpa hijauan dan growth promoter. Hasilnya adalah sebagai berikut : 1) sebanyak 20 ekor induk kambing mengalami birahi bersamaan menggunakan PGF2α sebesar 100%, Inseminasi buatan pada 10 ekor induk kambing tanpa super ovulasi mempunyai anak rata-rata 1,6 ekor sekelahiran, sedangkan Inseminasi buatan pada 10 ekor induk kambing dengan super ovulasi mempunyai anak rata-rata 3 ekor sekelahiran, 3) peningkatan berat badan kambing rata- rata 252,35 gram/ekor perhari.
3
Li, Jinyang, Sheng Tong, Farrukh Raza Amin, Habiba Khalid, Kai Chen, Xiaoguang Zhao, Jinling Cai e Demao Li. "Enhancing the Activity of a Self-Inducible Promoter in Escherichia coli through Saturation Mutation and High-Throughput Screening". Fermentation 9, n. 5 (13 maggio 2023): 468. http://dx.doi.org/10.3390/fermentation9050468.
Testo completoAbstract (sommario):
The use of self-inducible promoters is a promising strategy to address metabolic imbalances caused by overexpression. However, the low activity of natural self-inducible promoters hinders their widespread application. To overcome this limitation, we selected the fic promoter as a model promoter to create an enhanced self-inducible promoter library using saturation mutations and high-throughput screening. Sequence analysis revealed that these promoters share certain characteristics, including semi-conservation in the −35 hexamer, highly conserved cytosine in the −17 motif (compared to −13 for other promoters), and moderate A+T content between positions −33 and −18 in the spacer region. Additionally, the discriminator region of these promotors features high A+T content in the first five bases. We identified PficI-17, PficII-33, and PficIII-14 promoters as the optional promoters in the −35 hexamer, spacer region, and discriminator mutation libraries, respectively. These promotors were used as representatives to measure the specific fluorescence and OD600 nm dynamics in different media and to confirm their effect on the expression of different proteins, including egfp (enhanced green fluorescence protein) and rfp (red fluorescence protein). Overall, our findings provide valuable guidance for modifying promoters and developing a promoter library suitable for regulating target genes.
4
Shujaat, Muhammad, Abdul Wahab, Hilal Tayara e Kil To Chong. "pcPromoter-CNN: A CNN-Based Prediction and Classification of Promoters". Genes 11, n. 12 (21 dicembre 2020): 1529. http://dx.doi.org/10.3390/genes11121529.
Testo completoAbstract (sommario):
A promoter is a small region within the DNA structure that has an important role in initiating transcription of a specific gene in the genome. Different types of promoters are recognized by their different functions. Due to the importance of promoter functions, computational tools for the prediction and classification of a promoter are highly desired. Promoters resemble each other; therefore, their precise classification is an important challenge. In this study, we propose a convolutional neural network (CNN)-based tool, the pcPromoter-CNN, for application in the prediction of promotors and their classification into subclasses σ70, σ54, σ38, σ32, σ28 and σ24. This CNN-based tool uses a one-hot encoding scheme for promoter classification. The tools architecture was trained and tested on a benchmark dataset. To evaluate its classification performance, we used four evaluation metrics. The model exhibited notable improvement over that of existing state-of-the-art tools.
5
Mahoney, Michael E., e Daniel L. Wulff. "Mutations that Improve the pRE Promoter of Coliphage Lambda". Genetics 115, n. 4 (1 aprile 1987): 591–95. http://dx.doi.org/10.1093/genetics/115.4.591.
Testo completoAbstract (sommario):
ABSTRACT The dya5 mutation, a C→T change at position -43 of the λ pRE promoter, results in a twofold increase in pRE activity in vivo. Smaller increases in pRE activity are found for the dya2 mutation, a T→C change at position -1 of pRE, and the dya3 mutation, an A→G change at +5 of pRE· The mutant pRE promoters retain complete dependence on cII protein for activity. These observations argue, at least for pRE-like promoters, that promoter activities are influenced by nucleotide sequences at least eight nucleotides to the 5′-side of the conventional -35 region consensus sequence, and by nucleotide sequences near the start-site of transcription. Although Hawley and McClure (1983) found A·T pairs more frequently than G·TC pairs in the region of -40 to -45 of prokaryotic promoters, other mutations that change a G·TC pair to an A·T pair at positions -41, -44 and -45 of pRE do not result in increased promoter activity. We also found that a T→C change at position -42 results in a mild decrease in promoter activity. These observations argue that Ts at positions -42 and -43 of pRE are required for maximum promoter activity, but do not support the hypothesis that As and Ts in the -40 to -45 region generally lead to higher promoter activities.
6
George, Janet A., e Mary-Lou Pardue. "The Promoter of the Heterochromatic Drosophila Telomeric Retrotransposon, HeT-A, Is Active When Moved Into Euchromatic Locations". Genetics 163, n. 2 (1 febbraio 2003): 625–35. http://dx.doi.org/10.1093/genetics/163.2.625.
Testo completoAbstract (sommario):
Abstract The Drosophila telomeric retrotransposon, HeT-A, is found only in heterochromatin; therefore, its promoter must function in this chromatin environment. Studies of position effect variegation suggest that promoters of heterochromatic genes are very different from euchromatic promoters, but this idea has not been tested with isolated promoter sequences. The HeT-A promoter is the first heterochromatin promoter to be isolated and it is of interest to investigate its activity when removed from telomeric heterochromatin. This promoter was initially characterized by testing reporter constructs in transient transfection of cultured cells, an environment that may approximate its endogenous heterochromatin. We now report P-element-mediated transpositions of these constructs, testing the function of different parts of the putative promoter in euchromatin. Expression of endogenous HeT-A RNA shows marked developmental regulation and accumulates preferentially in replicating diploid tissues. HeT-A promoter constructs are active in all euchromatic locations tested and some display aspects of endogenous HeT-A stage- and cell-type expression programs. The activity of each promoter construct in euchromatic locations is also generally consistent with its activity in the transient transfection tests; a possibly significant exception is one sequence segment that appreciably enhanced activity in transient transfection but repressed promoter activity in euchromatin.
7
Voelkel-Meiman, K., e G. S. Roeder. "Gene conversion tracts stimulated by HOT1-promoted transcription are long and continuous." Genetics 126, n. 4 (1 dicembre 1990): 851–67. http://dx.doi.org/10.1093/genetics/126.4.851.
Testo completoAbstract (sommario):
Abstract The recombination-stimulating sequence, HOT1, corresponds to the promoter of transcription by yeast RNA polymerase I. The effect of HOT1 on mitotic interchromosomal recombination was examined in diploid strains carrying a heterozygous URA3 gene on chromosome III. The frequency of Ura- recombinants was increased 20-fold when HOT1 was inserted into the chromosome III copy marked with URA3, at a location 48 kbp centromere-proximal to URA3. Ura- recombinants were increased only 2-fold when HOT1 and URA3 were on opposite homologues. These results suggest that most HOT1-promoted Ura- recombinants result from gene conversion and that sequences on the HOT1-containing chromosome are preferentially converted. Characterization of Ura- recombinants isolated from strains carrying multiple markers on chromosome III indicates that HOT1-promoted gene conversion tracts are unusually long (often greater than 75 kbp) and almost always continuous. Furthermore, conversion tracts frequently extend to both sides of HOT1. We suggest that HOT1 promotes the formation of a double-strand break which is often followed by exonucleolytic digestion. Repair of the broken chromosome could then result from gap repair or from replicative repair primed only by the centromere-containing chromosomal fragment.
8
Davey, James A., e Corey J. Wilson. "Engineered signal-coupled inducible promoters: measuring the apparent RNA-polymerase resource budget". Nucleic Acids Research 48, n. 17 (5 settembre 2020): 9995–10012. http://dx.doi.org/10.1093/nar/gkaa734.
Testo completoAbstract (sommario):
Abstract Inducible promoters are a central regulatory component in synthetic biology, metabolic engineering, and protein production for laboratory and commercial uses. Many of these applications utilize two or more exogenous promoters, imposing a currently unquantifiable metabolic burden on the living system. Here, we engineered a collection of inducible promoters (regulated by LacI-based transcription factors) that maximize the free-state of endogenous RNA polymerase (RNAP). We leveraged this collection of inducible promotors to construct simple two-channel logical controls that enabled us to measure metabolic burden – as it relates to RNAP resource partitioning. The two-channel genetic circuits utilized sets of signal-coupled transcription factors that regulate cognate inducible promoters in a coordinated logical fashion. With this fundamental genetic architecture, we evaluated the performance of each inducible promoter as discrete operations, and as coupled systems to evaluate and quantify the effects of resource partitioning. Obtaining the ability to systematically and accurately measure the apparent RNA-polymerase resource budget will enable researchers to design more robust genetic circuits, with significantly higher fidelity. Moreover, this study presents a workflow that can be used to better understand how living systems adapt RNAP resources, via the complementary pairing of constitutive and regulated promoters that vary in strength.
9
Fandl, J. P., L. K. Thorner e S. W. Artz. "Mutations that affect transcription and cyclic AMP-CRP regulation of the adenylate cyclase gene (cya) of Salmonella typhimurium." Genetics 125, n. 4 (1 agosto 1990): 719–27. http://dx.doi.org/10.1093/genetics/125.4.719.
Testo completoAbstract (sommario):
Abstract We studied the expression of the cya promoter(s) in cya-lac fusion strains of Salmonella typhimurium and demonstrated cAMP receptor protein (CRP)-dependent repression by cAMP. Expression of cya was reduced about fourfold in cultures grown in acetate minimal medium as compared to cultures grown in glucose-6-phosphate minimal medium. Expression of cya was also reduced about fourfold by addition of 5 mM cAMP to cultures grown in glucose minimal medium. We constructed in vitro deletion and insertion mutations altering a major cya promoter (P2) and a putative CRP binding site overlapping P2. These mutations were recombined into the chromosome by allele replacement with M13mp::cya recombinant phages and the regulation of the mutant promoters was analyzed. A 4-bp deletion of the CRP binding site and a 4-bp insertion in this site nearly eliminated repression by cAMP. A mutant with the P2 promoter and the CRP binding site both deleted exhibited an 80% reduction in cya expression; the 20% residual expression was insensitive to cAMP repression. This mutant retained a Cya+ phenotype. Taken together, the results establish that the cya gene is transcribed from multiple promoters one of which, P2, is negatively regulated by the cAMP-CRP complex. Correction for the contribution to transcription by the cAMP-CRP nonregulated cya promoters indicates that the P2 promoter is repressed at least eightfold by cAMP-CRP.
10
Greener, A., S. M. Lehman e D. R. Helinski. "Promoters of the broad host range plasmid RK2: analysis of transcription (initiation) in five species of gram-negative bacteria." Genetics 130, n. 1 (1 gennaio 1992): 27–36. http://dx.doi.org/10.1093/genetics/130.1.27.
Testo completoAbstract (sommario):
Abstract A broad host range cloning vector was constructed, suitable for monitoring promoter activity in diverse Gram-negative bacteria. This vector, derived from plasmid RSF1010, utilized the firefly luciferase gene as the reporter, since the assay for its bioluminescent product is sensitive, and measurements can be made without background from the host. Twelve DNA fragments with promoter activity were obtained from broad host range plasmid RK2 and inserted into the RSF1010 derived vector. The relative luciferase activities were determined for these fragments in five species of Gram-negative bacteria. In addition, four promoters were analyzed by primer extension to locate transcriptional start sites in each host. The results show that several of the promoters vary substantially in relative strengths or utilize different transcriptional start sites in different bacteria. Other promoters exhibited similar activities and identical start sites in the five hosts examined.
11
Arkhipova, I. R. "Promoter elements in Drosophila melanogaster revealed by sequence analysis." Genetics 139, n. 3 (1 marzo 1995): 1359–69. http://dx.doi.org/10.1093/genetics/139.3.1359.
Testo completoAbstract (sommario):
Abstract A Drosophila Promoter Database containing 252 independent Drosophila melanogaster promoter entries has been compiled. The database and its subsets have been searched for overrepresented sequences. The analysis reveals that the proximal promoter region displays the most dramatic nucleotide sequence irregularities and exhibits a tripartite structure, consisting of TATA at -25/-30 bp, initiator (Inr) at +/- 5 bp and a novel class of downstream elements at +20/+30 bp from the RNA start site. These latter elements are also strand-specific. However, they differ from TATA and Inr in several aspects: (1) they are represented not by a single, but by multiple sequences, (2) they are shorter, (3) their position is less strictly fixed with respect to the RNA start site, (4) they emerge as a characteristic feature of Drosophila promoters and (5) some of them are strongly overrepresented in the TATA-less, but not TATA-containing, subset. About one-half of known Drosophila promoters can be classified as TATA-less. The overall sequence organization of the promoter region is characterized by an extended region with an increase in GC-content and a decrease in A, which contains a number of binding sites for Drosophila transcription factors.
12
Graña, D., T. Gardella e M. M. Susskind. "The effects of mutations in the ant promoter of phage P22 depend on context." Genetics 120, n. 2 (1 ottobre 1988): 319–27. http://dx.doi.org/10.1093/genetics/120.2.319.
Testo completoAbstract (sommario):
Abstract Recombination was used to construct 22 two- or three-way combinations of down- and up-mutations in Pant, a strong, near-consensus promoter of phage P22. The relative strengths of these promoters in vivo were assayed by fusing them to an ant/lacZ gene fusion and measuring beta-galactosidase levels produced by lysogens carrying the fusions on single-copy prophages. The results of these assays show that the magnitude of the effect of a promoter mutation can vary considerably when its context is changed by the presence of another mutation. In addition, as Pant approaches conformity with the consensus promoter sequence, the up-mutations decrease promoter strength, even though the same mutations increase promoter strength in the presence of a down-mutation. These context effects imply that individual consensus base pairs cannot be considered to contribute to promoter strength independently.
13
Inderbitzin, Anne, Yik Lim Kok, Lisa Jörimann, Audrey Kelley, Kathrin Neumann, Daniel Heinzer, Toni Cathomen e Karin J. Metzner. "HIV-1 promoter is gradually silenced when integrated into BACH2 in Jurkat T-cells". PeerJ 8 (24 novembre 2020): e10321. http://dx.doi.org/10.7717/peerj.10321.
Testo completoAbstract (sommario):
Background The persistence of the latent HIV-1 reservoir is a major obstacle to curing HIV-1 infection. HIV-1 integrates into the cellular genome and some targeted genomic loci are frequently detected in clonally expanded latently HIV-1 infected cells, for instance, the gene BTB domain and CNC homology 2 (BACH2). Methods We investigated HIV-1 promoter activity after integration into specific sites in BACH2 in Jurkat T-cells. The HIV-1-based vector LTatCL[M] contains two fluorophores: (1) Cerulean, which reports the activity of the HIV-1 promoter and (2) mCherry driven by a constitutive promotor and flanked by genetic insulators. This vector was inserted into introns 2 and 5 of BACH2 of Jurkat T-cells via CRISPR/Cas9 technology in the same and convergent transcriptional orientation of BACH2, and into the genomic safe harbour AAVS1. Single cell clones representing active (Cerulean+/mCherry+) and inactive (Cerulean–/mCherry+) HIV-1 promoters were characterised. Results Upon targeted integration of the 5.3 kb vector LTatCL[M] into BACH2, the HIV-1 promoter was gradually silenced as reflected by the decrease in Cerulean expression over a period of 162 days. Silenced HIV-1 promoters could be reactivated by TNF-α and Romidepsin. This observation was independent of the targeted intron and the transcriptional orientation. BACH2 mRNA and protein expression was not impaired by mono-allelic integration of LTatCL[M]. Conclusion Successful targeted integration of the HIV-1-based vector LTatCL[M] allows longitudinal analyses of HIV-1 promoter activity.
14
Liliana, Villao-Uzho, Chávez-Navarrete Tatiana, Pacheco-Coello Ricardo, Sánchez-Timm Eduardo e Santos-Ordóñez Efrén. "Plant Promoters: Their Identification, Characterization, and Role in Gene Regulation". Genes 14, n. 6 (6 giugno 2023): 1226. http://dx.doi.org/10.3390/genes14061226.
Testo completoAbstract (sommario):
One of the strategies to overcome diseases or abiotic stress in crops is the use of improved varieties. Genetic improvement could be accomplished through different methods, including conventional breeding, induced mutation, genetic transformation, or gene editing. The gene function and regulated expression through promoters are necessary for transgenic crops to improve specific traits. The variety of promoter sequences has increased in the generation of genetically modified crops because they could lead to the expression of the gene responsible for the improved trait in a specific manner. Therefore, the characterization of the promoter activity is necessary for the generation of biotechnological crops. That is why several analyses have focused on identifying and isolating promoters using techniques such as reverse transcriptase-polymerase chain reaction (RT-PCR), genetic libraries, cloning, and sequencing. Promoter analysis involves the plant genetic transformation method, a potent tool for determining the promoter activity and function of genes in plants, contributing to understanding gene regulation and plant development. Furthermore, the study of promoters that play a fundamental role in gene regulation is highly relevant. The study of regulation and development in transgenic organisms has made it possible to understand the benefits of directing gene expression in a temporal, spatial, and even controlled manner, confirming the great diversity of promoters discovered and developed. Therefore, promoters are a crucial tool in biotechnological processes to ensure the correct expression of a gene. This review highlights various types of promoters and their functionality in the generation of genetically modified crops.
15
Tang, Hongting, Yanling Wu, Jiliang Deng, Nanzhu Chen, Zhaohui Zheng, Yongjun Wei, Xiaozhou Luo e Jay D. Keasling. "Promoter Architecture and Promoter Engineering in Saccharomyces cerevisiae". Metabolites 10, n. 8 (6 agosto 2020): 320. http://dx.doi.org/10.3390/metabo10080320.
Testo completoAbstract (sommario):
Promoters play an essential role in the regulation of gene expression for fine-tuning genetic circuits and metabolic pathways in Saccharomyces cerevisiae (S. cerevisiae). However, native promoters in S. cerevisiae have several limitations which hinder their applications in metabolic engineering. These limitations include an inadequate number of well-characterized promoters, poor dynamic range, and insufficient orthogonality to endogenous regulations. Therefore, it is necessary to perform promoter engineering to create synthetic promoters with better properties. Here, we review recent advances related to promoter architecture, promoter engineering and synthetic promoter applications in S. cerevisiae. We also provide a perspective of future directions in this field with an emphasis on the recent advances of machine learning based promoter designs.
16
Christophides, George K., Ioannis Livadaras, Charalambos Savakis e Katia Komitopoulou. "Two Medfly Promoters That Have Originated by Recent Gene Duplication Drive Distinct Sex, Tissue and Temporal Expression Patterns". Genetics 156, n. 1 (1 settembre 2000): 173–82. http://dx.doi.org/10.1093/genetics/156.1.173.
Testo completoAbstract (sommario):
Abstract Genes encoding predominantly male-specific serum polypeptides (MSSPs) in the medfly Ceratitis capitata are members of a multigene family that are structurally similar to the genes encoding odorant binding proteins of insects. To study the transcriptional regulation of the genes MSSP-α2 and MSSP-β2, overlapping fragments of their promoters, containing the 5′ UTRs and 5′ flanking regions, were fused to the lacZ reporter gene and introduced into the medfly genome via Minos-mediated germline transformation. Transgenic flies were functionally assayed for β-galactosidase activity. Despite their extensive sequence similarity, the two gene promoters show distinct expression patterns of the reporter gene, consistent with previously reported evidence for analogous transcriptional activity of the corresponding endogenous genes. The MSSP-α2 promoter drives gene expression specifically in the fat body of the adult males, whereas the MSSP-β2 promoter directs gene expression in the midgut of both sexes. In contrast, similar transformation experiments in Drosophila melanogaster showed that both promoters drive the expression of the reporter gene in the midgut of adult flies of both sexes. Thus, the very same MSSP-α2 promoter fragment directs expression in the adult male fat body in Ceratitis, but in the midgut of both sexes in Drosophila. Our data suggest that through the evolution of the MSSP gene family a limited number of mutations that occurred within certain cis-acting elements, in combination with new medfly-specific trans-acting factors, endowed these recently duplicated genes with distinct sex-, tissue-, and temporal-specific expression patterns.
17
Wu, Limin, Aliaa El-Mezawy e Saleh Shah. "Isolation and evaluation of three novel native promoters in Brassica napus". Botany 91, n. 6 (giugno 2013): 414–19. http://dx.doi.org/10.1139/cjb-2012-0245.
Testo completoAbstract (sommario):
To provide effective and specific native promoters for canola (Brassica napus L.) genetic modification, three promoters were isolated by genome walking from this species. These three promoters were fused to the uidA reporter gene (GUS) and were independently used to generate populations of transgenic canola plants. Plants transformed with BnPGPro-GUS (B. napus putative germin promoter) exhibited GUS activity in all the tissues tested at a level comparable to those transformed with CaMV35 S promoter. This indicates that BnPGPro may serve as a native constitutive promoter for canola. The other two promoters, BnPro3-GUS and BnPro5-GUS (B. napus, promoter 3 and 5), exhibited GUS activity in various tissues. None of these two promoters expressed in embryo, however. These novel Brassica native promoters can be used to modify canola genes for various purposes.
18
Southworth, Jeffrey W., e James A. Kennison. "Transvection and Silencing of the Scr Homeotic Gene of Drosophila melanogaster". Genetics 161, n. 2 (1 giugno 2002): 733–46. http://dx.doi.org/10.1093/genetics/161.2.733.
Testo completoAbstract (sommario):
Abstract The Sex combs reduced (Scr) gene specifies the identities of the labial and first thoracic segments in Drosophila melanogaster. In imaginal cells, some Scr mutations allow cis-regulatory elements on one chromosome to stimulate expression of the promoter on the homolog, a phenomenon that was named transvection by Ed Lewis in 1954. Transvection at the Scr gene is blocked by rearrangements that disrupt pairing, but is zeste independent. Silencing of the Scr gene in the second and third thoracic segments, which requires the Polycomb group proteins, is disrupted by most chromosomal aberrations within the Scr gene. Some chromosomal aberrations completely derepress Scr even in the presence of normal levels of all Polycomb group proteins. On the basis of the pattern of chromosomal aberrations that disrupt Scr gene silencing, we propose a model in which two cis-regulatory elements interact to stabilize silencing of any promoter or cis-regulatory element physically between them. This model also explains the anomalous behavior of the Scx allele of the flanking homeotic gene, Antennapedia. This allele, which is associated with an insertion near the Antennapedia P1 promoter, inactivates the Antennapedia P1 and P2 promoters in cis and derepresses the Scr promoters both in cis and on the homologous chromosome.
19
Miki, Shunichiro, Tomoyuki Koga, Alison Parisian e Frank Furnari. "TMOD-09. MODELING TERT PROMOTER MUTATION IN ISOGENIC NEXT GENERATION GBM MODELS". Neuro-Oncology 21, Supplement_6 (novembre 2019): vi264. http://dx.doi.org/10.1093/neuonc/noz175.1108.
Testo completoAbstract (sommario):
Abstract Telomere reverse transcriptase (TERT) promotor mutations increase TERT expression and are known to be the most common single genetic abnormality in Glioblastoma (GBM). Recent studies have revealed this mutation to be an early event in gliomagenesis but it is detected subclonally in a considerable number of cases. Due to the long telomere length and TERT expression in somatic cells of mice, genetically engineered mouse models of GBM do not address the function of this mutation and thus an ideal heterozygous TERT promotor mutation model is yet to be established. Recently, our lab established strategies for generating GBM models using neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (hiPSCs) that have been CRISPR/Cas9 engineered with different combinations of authentic GBM-related genetic drivers which can be used for introduction of mutant TERT promotor (MTP) sequence and examination of its function. We obtained multiple heterozygous MTP hiPS clones in the context of GBM genetic alterations, CDKN2A/B and PTEN deletion, using a two-step targeting approach. Two single-strand guide RNAs (sgRNAs) were used to delete the TERT promotor and a portion of exon 1 followed by repair of the locus with wild type TERT promoter (WTP) and MTP sequence. Following promoter reconstitution, TERT promotor function was tested in iPSCs, NPCs, and astrocytes. This analysis revealed successful TERT expression silencing in WTP astrocytes. Furthermore, orthotopic injection of WTP and MTP NPCs into immunodeficient mice formed pathologically similar GBM-like tumors in same period of time. TERT expression was only detected in cell lines derived from MTP tumors, indicating TERT promotor mutation has little impact in initiation of tumor formation from NPCs, explaining its subclonality in patient tumors. Our model enables further research on MTP function and dependency during gliomagenesis.
20
Dong, Yuankai, S. V. Satya Prakash Avva, Mukesh Maharjan, Janice Jacobi e Craig M. Hart. "Promoter-Proximal Chromatin Domain Insulator Protein BEAF Mediates Local and Long-Range Communication with a Transcription Factor and Directly Activates a Housekeeping Promoter in Drosophila". Genetics 215, n. 1 (16 marzo 2020): 89–101. http://dx.doi.org/10.1534/genetics.120.303144.
Testo completoAbstract (sommario):
BEAF (Boundary Element-Associated Factor) was originally identified as a Drosophila melanogaster chromatin domain insulator-binding protein, suggesting a role in gene regulation through chromatin organization and dynamics. Genome-wide mapping found that BEAF usually binds near transcription start sites, often of housekeeping genes, suggesting a role in promoter function. This would be a nontraditional role for an insulator-binding protein. To gain insight into molecular mechanisms of BEAF function, we identified interacting proteins using yeast two-hybrid assays. Here, we focus on the transcription factor Serendipity δ (Sry-δ). Interactions were confirmed in pull-down experiments using bacterially expressed proteins, by bimolecular fluorescence complementation, and in a genetic assay in transgenic flies. Sry-δ interacted with promoter-proximal BEAF both when bound to DNA adjacent to BEAF or > 2-kb upstream to activate a reporter gene in transient transfection experiments. The interaction between BEAF and Sry-δ was detected using both a minimal developmental promoter (y) and a housekeeping promoter (RpS12), while BEAF alone strongly activated the housekeeping promoter. These two functions for BEAF implicate it in playing a direct role in gene regulation at hundreds of BEAF-associated promoters.
21
Efremova, Larisa N., Svetlana R. Strelnikova, Guzel R. Gazizova, Elena A. Minkina e Roman A. Komakhin. "A Synthetic Strong and Constitutive Promoter Derived from the Stellaria media pro-SmAMP1 and pro-SmAMP2 Promoters for Effective Transgene Expression in Plants". Genes 11, n. 12 (26 novembre 2020): 1407. http://dx.doi.org/10.3390/genes11121407.
Testo completoAbstract (sommario):
Synthetic promoters are vital for genetic engineering-based strategies for crop improvement, but effective methodologies for their creation and systematic testing are lacking. We report here on the comparative analysis of the promoters pro-SmAMP1 and pro-SmAMP2 from Stellaria media ANTIMICROBIAL PEPTIDE1 (AMP1) and ANTIMICROBIAL PEPTIDE2 (AMP2). These promoters are more effective than the well-known Cauliflower mosaic virus 35S promoter. Although these promoters share about 94% identity, the pro-SmAMP1 promoter demonstrated stronger transient expression of a reporter gene in Agrobacterium infiltration of Nicotiana benthamiana leaves, while the pro-SmAMP2 promoter was more effective for the selection of transgenic tobacco (Nicotiana tabacum) cells when driving a selectable marker. Using the cap analysis of gene expression method, we detected no differences in the structure of the transcription start sites for either promoter in transgenic plants. For both promoters, we used fine-scale deletion analysis to identify 160 bp-long sequences that retain the unique properties of each promoter. With the use of chimeric promoters and directed mutagenesis, we demonstrated that the superiority of the pro-SmAMP1 promoter for Agrobacterium-mediated infiltration is caused by the proline-inducible ACTCAT cis-element strictly positioned relative to the TATA box in the core promoter. Surprisingly, the ACTCAT cis-element not only activated but also suppressed the efficiency of the pro-SmAMP1 promoter under proline stress. The absence of the ACTCAT cis-element and CAANNNNATC motif (negative regulator) in the pro-SmAMP2 promoter provided a more constitutive gene expression profile and better selection of transgenic cells on selective medium. We created a new synthetic promoter that enjoys high effectiveness both in transient expression and in selection of transgenic cells. Intact promoters with differing properties and high degrees of sequence identity may thus be used as a basis for the creation of new synthetic promoters for precise and coordinated gene expression.
22
Daegelen, P., e E. Brody. "The rIIA gene of bacteriophage T4. II. Regulation of its messenger RNA synthesis." Genetics 125, n. 2 (1 giugno 1990): 249–60. http://dx.doi.org/10.1093/genetics/125.2.249.
Testo completoAbstract (sommario):
Abstract When the rII genes are first introduced into cells which had been previously infected by T4 phage deleted for these genes, the kinetics of synthesis of rIIA and rIIB RNA are rapid and identical. We show that this rapid synthesis depends on a functional motA gene for rIIB, but not for rIIA, RNA synthesis. By primer-extension mapping of T4 messenger RNA, we find three promoters close to the rIIA gene. One of them is an early promoter just before the rIIA.1 gene; it is used under all conditions tested. Another is in the coding portion of the rIIA.1 gene; it is weak, primarily because of a 19-bp spacing between the -10 and -35 elements, and its use is stimulated by T4 functions. The third is a motA-dependent (middle) promoter which has an unusual CCCGCTT box at -33. We present results which suggest that none of these promoters is likely to be the site at which the motB and motC gene products exercise their major influence on rIIA RNA synthesis.
23
Khan, Ahamed, Noohi Nasim, Baveesh Pudhuvai, Bhupendra Koul, Santosh Kumar Upadhyay, Lini Sethi e Nrisingha Dey. "Plant Synthetic Promoters: Advancement and Prospective". Agriculture 13, n. 2 (26 gennaio 2023): 298. http://dx.doi.org/10.3390/agriculture13020298.
Testo completoAbstract (sommario):
Native/endogenous promoters have several fundamental limitations in terms of their size, Cis-elements distribution/patterning, and mode of induction, which is ultimately reflected in their insufficient transcriptional activity. Several customized synthetic promoters were designed and tested in plants during the past decade to circumvent such constraints. Such synthetic promoters have a built-in capacity to drive the expression of the foreign genes at their maximum amplitude in plant orthologous systems. The basic structure and function of the promoter has been discussed in this review, with emphasis on the role of the Cis-element in regulating gene expression. In addition to this, the necessity of synthetic promoters in the arena of plant biology has been highlighted. This review also provides explicit information on the two major approaches for developing plant-based synthetic promoters: the conventional approach (by utilizing the basic knowledge of promoter structure and Cis-trans interaction) and the advancement in gene editing technology. The success of plant genetic manipulation relies on the promoter efficiency and the expression level of the transgene. Therefore, advancements in the field of synthetic promoters has enormous potential in genetic engineering-mediated crop improvement.
24
Okkema, P. G., S. W. Harrison, V. Plunger, A. Aryana e A. Fire. "Sequence requirements for myosin gene expression and regulation in Caenorhabditis elegans." Genetics 135, n. 2 (1 ottobre 1993): 385–404. http://dx.doi.org/10.1093/genetics/135.2.385.
Testo completoAbstract (sommario):
Abstract Four Caenorhabditis elegans genes encode muscle-type specific myosin heavy chain isoforms: myo-1 and myo-2 are expressed in the pharyngeal muscles; unc-54 and myo-3 are expressed in body wall muscles. We have used transformation-rescue and lacZ fusion assays to determine sequence requirements for regulated myosin gene expression during development. Multiple tissue-specific activation elements are present for all four genes. For each of the four genes, sequences upstream of the coding region are tissue-specific promoters, as shown by their ability to drive expression of a reporter gene (lacZ) in the appropriate muscle type. Each gene contains at least one additional tissue-specific regulatory element, as defined by the ability to enhance expression of a heterologous promoter in the appropriate muscle type. In rescue experiments with unc-54, two further requirements apparently independent of tissue specificity were found: sequences within the 3' non-coding region are essential for activity while an intron near the 5' end augments expression levels. The general intron stimulation is apparently independent of intron sequence, indicating a mechanistic effect of splicing. To further characterize the myosin gene promoters and to examine the types of enhancer sequences in the genome, we have initiated a screen of C. elegans genomic DNA for fragments capable of enhancing the myo-2 promoter. The properties of enhancers recovered from this screen suggest that the promoter is limited to muscle cells in its ability to respond to enhancers.
25
Atkinson, P. W., L. E. Mills, W. T. Starmer e D. T. Sullivan. "Structure and evolution of the Adh genes of Drosophila mojavensis." Genetics 120, n. 3 (1 novembre 1988): 713–23. http://dx.doi.org/10.1093/genetics/120.3.713.
Testo completoAbstract (sommario):
Abstract The nucleotide sequence of the Adh region of Drosophila mojavensis has been completed and the region found to contain a pseudogene, Adh-2 and Adh-1 arranged in that order. Comparison of the sequence divergence of these genes to one another and to the Adh region of Drosophila mulleri and other species has allowed the development of a model for the evolution of the duplication of the Adh genes. There have been two major events. An initial duplication of an Adh gene whose dual promoter structure was similar to Drosophila melanogaster, resulted in a species with two Adh genes, one of which may have had only a proximal promoter. A second duplication of this gene generated an Adh region containing three genes. It is proposed that one of these is the ancestral gene having dual promoters, while the other two possess only proximal promoters. Subsequent events have resulted in both a change in the regulation of Adh-2 such that it is expressed as if it had a "distal" type promoter and the mutational inactivation of the most upstream gene resulting in the creation of a pseudogene. The sequence of the D. mojavensis Adh region has also revealed the presence of an element which is composed of juxtaposed inverted imperfectly repeated elements. There is a surprising and not fully explainable strong similarity of the nucleotide sequence of the 5' flanking region of the pseudogene in D. mojavensis and D. mulleri.
26
Thorner, L. K., J. P. Fandl e S. W. Artz. "Analysis of sequence elements important for expression and regulation of the adenylate cyclase gene (cya) of Salmonella typhimurium." Genetics 125, n. 4 (1 agosto 1990): 709–17. http://dx.doi.org/10.1093/genetics/125.4.709.
Testo completoAbstract (sommario):
Abstract We determined the nucleotide sequence of the regulatory region of the cya gene of Salmonella typhimurium. A set of nested BAL-31 deletions originating upstream of the promoter/regulatory region and extending into the cya structural gene was constructed in M13mp::cya phages and was tested for complementation of a chromosomal cya deletion mutation. BAL-31 deletion mutants unable to complement cya localized the major cya promoter. The synthetic tac promoter was inserted upstream of the BAL-31 deletions so that expression of cya was dependent on transcription from tac. Those tac derivative phages unable to complement cya localized the translation initiation region. The cya DNA sequence revealed at least three potential promoters capable of transcribing cya, with a CRP binding site straddling the-10 hexamer of the promoter proximal to the structural gene. The leader RNA sequence initiated at the latter promoter is approximately 140 bases long and includes a region that may form a stable secondary structure (delta G = -23.8 kcal). There exist two possible in-frame translation start points, one of which is TTG and the other of which is ATG. The sequence of the S. typhimurium regulatory region was compared with that reported for Escherichia coli.
27
Xu, Mengqiong, Sisi Xia, Mei Wang, Xiaolian Liu, Xin Li, Weijie Chen, Yaohao Wang et al. "Enzymatic independent role of sphingosine kinase 2 in regulating the expression of type I interferon during influenza A virus infection". PLOS Pathogens 18, n. 9 (7 settembre 2022): e1010794. http://dx.doi.org/10.1371/journal.ppat.1010794.
Testo completoAbstract (sommario):
Influenza virus has the ability to circumvent host innate immune system through regulating certain host factors for its effective propagation. However, the detailed mechanism is still not fully understood. Here, we report that a host sphingolipid metabolism-related factor, sphingosine kinase 2 (SPHK2), upregulated during influenza A virus (IAV) infection, promotes IAV infection in an enzymatic independent manner. The enhancement of the virus replication is not abolished in the catalytic-incompetent SPHK2 (G212E) overexpressing cells. Intriguingly, the sphingosine-1-phosphate (S1P) related factor HDAC1 also plays a crucial role in SPHK2-mediated IAV infection. We found that SPHK2 cannot facilitate IAV infection in HDAC1 deficient cells. More importantly, SPHK2 overexpression diminishes the IFN-β promoter activity upon IAV infection, resulting in the suppression of type I IFN signaling. Furthermore, ChIP-qPCR assay revealed that SPHK2 interacts with IFN-β promoter through the binding of demethylase TET3, but not with the other promoters regulated by TET3, such as TGF-β1 and IL6 promoters. The specific regulation of SPHK2 on IFN-β promoter through TET3 can in turn recruit HDAC1 to the IFN-β promoter, enhancing the deacetylation of IFN-β promoter, therefore leading to the inhibition of IFN-β transcription. These findings reveal an enzymatic independent mechanism on host SPHK2, which associates with TET3 and HDAC1 to negatively regulate type I IFN expression and thus facilitates IAV propagation.
28
Chiang, L. W., e M. M. Howe. "Mutational analysis of a C-dependent late promoter of bacteriophage Mu." Genetics 135, n. 3 (1 novembre 1993): 619–29. http://dx.doi.org/10.1093/genetics/135.3.619.
Testo completoAbstract (sommario):
Abstract Late transcription of bacteriophage Mu initiates at four promoters, P(lys), PI, PP and Pmom, and requires the Mu C protein and the host RNA polymerase. Promoter-containing DNA fragments extending approximately 200 bp upstream and downstream of the 5' starts of the lys, I and P transcripts were cloned into a multicopy lacZ-expression plasmid. Promoter activity, assayed by beta-galactosidase expression, was determined under two different conditions: (1) with C provided from a compatible plasmid in the absence of other Mu factors and (2) with C provided from an induced Mu prophage. beta-galactosidase activities were greatest for P(lys), intermediate for PI, and lowest for PP. Similar analysis of plasmids containing nested sets of deletions removing 5' or 3' sequences of P(lys) demonstrated that a 68-bp region was sufficient for full activity. Point mutations were generated within the 68-bp region by mutagenic oligonucleotide-directed PCR (Mod-PCR). Properties of the lys promoter mutants indicated that, in addition to the -10 region, a 19-bp region from -52 to -34 containing the C footprint is required for C-dependent promoter activity.
29
He, Kevin, S. M. Ali Hosseini Rad, Aarati Poudel e Alexander Donald McLellan. "Compact Bidirectional Promoters for Dual-Gene Expression in a Sleeping Beauty Transposon". International Journal of Molecular Sciences 21, n. 23 (4 dicembre 2020): 9256. http://dx.doi.org/10.3390/ijms21239256.
Testo completoAbstract (sommario):
Promoter choice is an essential consideration for transgene expression in gene therapy. The expression of multiple genes requires ribosomal entry or skip sites, or the use of multiple promoters. Promoter systems comprised of two separate, divergent promoters may significantly increase the size of genetic cassettes intended for use in gene therapy. However, an alternative approach is to use a single, compact, bidirectional promoter. We identified strong and stable bidirectional activity of the RPBSA synthetic promoter comprised of a fragment of the human Rpl13a promoter, together with additional intron/exon structures. The Rpl13a-based promoter drove long-term bidirectional activity of fluorescent proteins. Similar results were obtained for the EF1-α and LMP2/TAP1 promoters. However, in a lentiviral vector, the divergent bidirectional systems failed to produce sufficient titres to translate into an expression system for dual chimeric antigen receptor (CAR) expression. Although bidirectional promoters show excellent applicability to drive short RNA in Sleeping Beauty transposon systems, their possible use in the lentiviral applications requiring longer and more complex RNA, such as dual-CAR cassettes, is limited.
30
Wang, Ye, Haochen Wang, Lei Wei, Shuailin Li, Liyang Liu e Xiaowo Wang. "Synthetic promoter design in Escherichia coli based on a deep generative network". Nucleic Acids Research 48, n. 12 (19 maggio 2020): 6403–12. http://dx.doi.org/10.1093/nar/gkaa325.
Testo completoAbstract (sommario):
Abstract Promoter design remains one of the most important considerations in metabolic engineering and synthetic biology applications. Theoretically, there are 450 possible sequences for a 50-nt promoter, of which naturally occurring promoters make up only a small subset. To explore the vast number of potential sequences, we report a novel AI-based framework for de novo promoter design in Escherichia coli. The model, which was guided by sequence features learned from natural promoters, could capture interactions between nucleotides at different positions and design novel synthetic promoters in silico. We combined a deep generative model that guides the search for artificial sequences with a predictive model to preselect the most promising promoters. The AI-designed promoters were optimized based on the promoter activity in E. coli and the predictive model. After two rounds of optimization, up to 70.8% of the AI-designed promoters were experimentally demonstrated to be functional, and few of them shared significant sequence similarity with the E. coli genome. Our work provided an end-to-end approach to the de novo design of novel promoter elements, indicating the potential to apply deep learning methods to de novo genetic element design.
31
Kashkin, K. N., I. P. Chernov, E. A. Stukacheva, E. P. Kopantzev, G. S. Monastyrskaya, N. Ya Uspenskaya e E. D. Sverdlov. "Cancer Specificity of Promoters of the Genes Involved in Cell Proliferation Control". Acta Naturae 5, n. 3 (15 settembre 2013): 79–83. http://dx.doi.org/10.32607/20758251-2013-5-3-79-83.
Testo completoAbstract (sommario):
Core promoters with adjacent regions of the human genes CDC6, POLD1, CKS1B, MCM2, and PLK1 were cloned into a pGL3 vector in front of the Photinus pyrails gene Luc in order to study the tumor specificity of the promoters. The cloned promoters were compared in their ability to direct luciferase expression in different human cancer cells and in normal fibroblasts. The cancer-specific promoter BIRC5 and non-specific CMV immediately early gene promoter were used for comparison. All cloned promoters were shown to be substantially more active in cancer cells than in fibroblasts, while the PLK1 promoter was the most cancer-specific and promising one. The specificity of the promoters to cancer cells descended in the series PLK1, CKS1B, POLD1, MCM2, and CDC6. The bidirectional activity of the cloned CKS1B promoter was demonstrated. It apparently directs the expression of the SHC1 gene, which is located in a head-to-head position to the CKS1B gene in the human genome. This feature should be taken into account in future use of the CKS1B promoter. The cloned promoters may be used in artificial genetic constructions for cancer gene therapy.
32
Feng, Xiaofan, e Mario Andrea Marchisio. "Saccharomyces cerevisiae Promoter Engineering before and during the Synthetic Biology Era". Biology 10, n. 6 (6 giugno 2021): 504. http://dx.doi.org/10.3390/biology10060504.
Testo completoAbstract (sommario):
Synthetic gene circuits are made of DNA sequences, referred to as transcription units, that communicate by exchanging proteins or RNA molecules. Proteins are, mostly, transcription factors that bind promoter sequences to modulate the expression of other molecules. Promoters are, therefore, key components in genetic circuits. In this review, we focus our attention on the construction of artificial promoters for the yeast S. cerevisiae, a popular chassis for gene circuits. We describe the initial techniques and achievements in promoter engineering that predated the start of the Synthetic Biology epoch of about 20 years. We present the main applications of synthetic promoters built via different methods and discuss the latest innovations in the wet-lab engineering of novel promoter sequences.
33
Blazeck, John, Leqian Liu, Heidi Redden e Hal Alper. "Tuning Gene Expression in Yarrowia lipolytica by a Hybrid Promoter Approach". Applied and Environmental Microbiology 77, n. 22 (16 settembre 2011): 7905–14. http://dx.doi.org/10.1128/aem.05763-11.
Testo completoAbstract (sommario):
ABSTRACTThe development of strong and tunable promoter elements is necessary to enable metabolic and pathway engineering applications for any host organism. Here, we have expanded and generalized a hybrid promoter approach to produce libraries of high-expressing, tunable promoters in the nonconventional yeastYarrowia lipolytica. These synthetic promoters are comprised of two modular components: the enhancer element and the core promoter element. By exploiting this basic promoter architecture, we have overcome native expression limitations and provided a strategy for both increasing the native promoter capacity and producing libraries for tunable gene expression in a cellular system with ill-defined genetic tools. In doing so, this work has created the strongest promoters ever reported forY. lipolytica. Furthermore, we have characterized these promoters at the single-cell level through the use of a developed fluorescence-based assay as well as at the transcriptional and whole-cell levels. The resulting promoter libraries exhibited a range of more than 400-fold in terms of mRNA levels, and the strongest promoters in this set had 8-fold-higher fluorescence levels than those of typically used endogenous promoters. These results suggest that promoters inY. lipolyticaare enhancer limited and that this limitation can be partially or fully alleviated through the addition of tandem copies of upstream activation sequences (UASs). Finally, this work illustrates that tandem copies of UAS regions can serve as synthetic transcriptional amplifiers that may be generically used to increase the expression levels of promoters.
34
Maharjan, Mukesh, J. Keller McKowen e Craig M. Hart. "Overlapping but Distinct Sequences Play Roles in the Insulator and Promoter Activities of the Drosophila BEAF-Dependent scs’ Insulator". Genetics 215, n. 4 (17 giugno 2020): 1003–12. http://dx.doi.org/10.1534/genetics.120.303344.
Testo completoAbstract (sommario):
Chromatin domain insulators are thought to help partition the genome into genetic units called topologically associating domains (TADs). In Drosophila, TADs are often separated by inter-TAD regions containing active housekeeping genes and associated insulator binding proteins. This raises the question of whether insulator binding proteins are involved primarily in chromosomal TAD architecture or gene activation, or if these two activities are linked. The Boundary Element-Associated Factor of 32 kDa (BEAF-32, or BEAF for short) is usually found in inter-TADs. BEAF was discovered based on binding to the scs’ insulator, and is important for the insulator activity of scs’ and other BEAF binding sites. There are divergent promoters in scs’ with a BEAF binding site by each. Here, we dissect the scs’ insulator to identify DNA sequences important for insulator and promoter activity, focusing on the half of scs’ with a high affinity BEAF binding site. We find that the BEAF binding site is important for both insulator and promoter activity, as is another sequence we refer to as LS4. Aside from that, different sequences play roles in insulator and promoter activity. So while there is overlap and BEAF is important for both, insulator and promoter activity can be separated.
35
Hong, Clarice K. Y., e Barak A. Cohen. "Genomic environments scale the activities of diverse core promoters". Genome Research 32, n. 1 (27 dicembre 2021): 85–96. http://dx.doi.org/10.1101/gr.276025.121.
Testo completoAbstract (sommario):
A classical model of gene regulation is that enhancers provide specificity whereas core promoters provide a modular site for the assembly of the basal transcriptional machinery. However, examples of core promoter specificity have led to an alternate hypothesis in which specificity is achieved by core promoters with different sequence motifs that respond differently to genomic environments containing different enhancers and chromatin landscapes. To distinguish between these models, we measured the activities of hundreds of diverse core promoters in four different genomic locations and, in a complementary experiment, six different core promoters at thousands of locations across the genome. Although genomic locations had large effects on expression, the intrinsic activities of different classes of promoters were preserved across genomic locations, suggesting that core promoters are modular regulatory elements whose activities are independently scaled up or down by different genomic locations. This scaling of promoter activities is nonlinear and depends on the genomic location and the strength of the core promoter. Our results support the classical model of regulation in which diverse core promoter motifs set the intrinsic strengths of core promoters, which are then amplified or dampened by the activities of their genomic environments.
36
Kreidberg, J. A., e T. J. Kelly. "Genetic analysis of the human thymidine kinase gene promoter". Molecular and Cellular Biology 6, n. 8 (agosto 1986): 2903–9. http://dx.doi.org/10.1128/mcb.6.8.2903-2909.1986.
Testo completoAbstract (sommario):
The promoter of the human thymidine kinase gene was defined by DNA sequence and genetic analyses. Mutant plasmids with deletions extending into the promoter region from both the 5' and 3' directions were constructed. The mutants were tested in a gene transfer system for the ability to transform TK- cells to the TK+ phenotype. This analysis delimited the functional promoter to within an 83-base-pair region upstream of the mRNA cap site. This region contains sequences common to other eucaryotic promoters including G X C-rich hexanucleotides, a CAAT box, and an A X T-rich region. The CAAT box is in an inverted orientation and is part of a 9-base-pair sequence repeated twice in the promoter region. Comparison of the genomic sequence with the cDNA sequence defined the first exon of the thymidine kinase gene.
37
Kreidberg, J. A., e T. J. Kelly. "Genetic analysis of the human thymidine kinase gene promoter." Molecular and Cellular Biology 6, n. 8 (agosto 1986): 2903–9. http://dx.doi.org/10.1128/mcb.6.8.2903.
Testo completoAbstract (sommario):
The promoter of the human thymidine kinase gene was defined by DNA sequence and genetic analyses. Mutant plasmids with deletions extending into the promoter region from both the 5' and 3' directions were constructed. The mutants were tested in a gene transfer system for the ability to transform TK- cells to the TK+ phenotype. This analysis delimited the functional promoter to within an 83-base-pair region upstream of the mRNA cap site. This region contains sequences common to other eucaryotic promoters including G X C-rich hexanucleotides, a CAAT box, and an A X T-rich region. The CAAT box is in an inverted orientation and is part of a 9-base-pair sequence repeated twice in the promoter region. Comparison of the genomic sequence with the cDNA sequence defined the first exon of the thymidine kinase gene.
38
Harr, Michael W., Xinyang Zhao, Mary Klein, Fan Liu, Megan Hatlen e Stephen Nimer. "JAK2 V617F, PRMT5, and GATA-1 Form a Regulatory Loop That Controls Autophagy Via Effects on ATG3 Gene Expression". Blood 118, n. 21 (18 novembre 2011): 2822. http://dx.doi.org/10.1182/blood.v118.21.2822.2822.
Testo completoAbstract (sommario):
Abstract Abstract 2822 The JAK2 V617F mutation is the most prevalent genetic abnormality in Philadelphia chromosome negative myeloproliferative neoplasia. We recently discovered that JAK2 V617F phosphorylates and inactivates PRMT5, a type II arginine methyltransferase that promotes transcriptional repression. To evaluate the PRMT5 dependent and independent effects of JAK2 V617F, we performed microarray analysis of human erythroleukemia cells (homozygous for JAK2 V617F) treated with a JAK2 inhibitor or transduced with PRMT5 shRNA. We discovered 5 autophagy-related genes (ATG3, ATG7, ATG2B, ATG5, and ATG16L1) that were positively regulated by JAK2 V617F, but negatively regulated by PRMT5. PRMT5 was bound to the promoters of these genes and binding was enhanced when PRMT5 was re-activated by a JAK2 inhibitor. Sequence analysis of ATG promoters identified at least one potential GATA-1 binding site in each ATG gene. Yet, knock-down of GATA-1 decreased the expression of ATG3 (by approximately 50%), but not the other ATG genes. ChIP analysis identified binding of both GATA-1 and PRMT5 to the ATG3 promoter, which suggests that ATG3 is transcriptionally induced by GATA-1 when PRMT5 is inactivated by JAK2 V617F. But when PRMT5 was active, it was bound to the GATA-1 promoter and ATG3 expression was downregulated. As JAK2 V617F (but not wild type) induces ATG3 expression and autophagy in AML cells and ATG3 levels are elevated in CD34+ cells from polycythemia vera patients, we conclude that JAK2 V617F promotes autophagy by inactivating PRMT5 and promoting GATA-1-dependent transcription of ATG3. Targeting autophagy with JAK2 inhibitors or other agents may be efficacious in the treatment of myeloproliferative neoplasia. Disclosures: No relevant conflicts of interest to declare.
39
Biswas, Debabrata, Yaxin Yu, Matthew Prall, Tim Formosa e David J. Stillman. "The Yeast FACT Complex Has a Role in Transcriptional Initiation". Molecular and Cellular Biology 25, n. 14 (luglio 2005): 5812–22. http://dx.doi.org/10.1128/mcb.25.14.5812-5822.2005.
Testo completoAbstract (sommario):
ABSTRACT A crucial step in eukaryotic transcriptional initiation is recognition of the promoter TATA by the TATA-binding protein (TBP), which then allows TFIIA and TFIIB to be recruited. However, nucleosomes block the interaction between TBP and DNA. We show that the yeast FACT complex (yFACT) promotes TBP binding to a TATA box in chromatin both in vivo and in vitro. The SPT16 gene encodes a subunit of yFACT, and we show that certain spt16 mutations are synthetically lethal with TBP mutants. Some of these genetic defects can be suppressed by TFIIA overexpression, strongly suggesting a role for yFACT in TBP-TFIIA complex formation in vivo. Mutations in the TOA2 subunit of TFIIA that disrupt TBP-TFIIA complex formation in vitro are also synthetically lethal with spt16. In some cases this spt16 toa2 lethality is suppressed by overexpression of TBP or the Nhp6 architectural transcription factor that is also a component of yFACT. The Spt3 protein in the SAGA complex has been shown to regulate TBP binding at certain promoters, and we show that some spt16 phenotypes can be suppressed by spt3 mutations. Chromatin immunoprecipitations show TBP binding to promoters is reduced in single spt16 and spt3 mutants but increases in the spt16 spt3 double mutant, reflecting the mutual suppression seen in the genetic assays. Finally, in vitro studies show that yFACT promotes TBP binding to a TATA sequence within a reconstituted nucleosome in a TFIIA-dependent manner. Thus, yFACT functions in establishing transcription initiation complexes in addition to the previously described role in elongation.
40
Morcillo, Patrick, Christina Rosen e Dale Dorsett. "Genes Regulating the Remote Wing Margin Enhancer in the Drosophila cut Locus". Genetics 144, n. 3 (1 novembre 1996): 1143–54. http://dx.doi.org/10.1093/genetics/144.3.1143.
Testo completoAbstract (sommario):
Abstract The mechanisms that allow enhancers to activate promoters from thousands of base pairs away are disrupted by the suppressor of Hairy-wing protein (SUHW) of Drosophila. SUHW binds a DNA sequence in the gypsy retrotransposon and prevents enhancers promoter-distal to a gypsy insertion in a gene from activating without affecting promoter-proximal enhancers. Several observations indicate that SUHW does not affect enhancer-binding activators. Instead, SUHW may interfere with factors that structurally facilitate interactions between an enhancer and promoter. To identify putative enhancer facilitators, a screen for mutations that reduce activity of the remote wing margin enhancer in the cut gene was performed. Mutations in scalloped, mastermind, and a previously unknown gene, Chip, were isolated. A TEA DNA-binding domain in the Scalloped protein binds the wing margin enhancer. Interactions between scalloped, mastermind and Chip mutations indicate that mastermind and Chip act synergistically with scalloped to regulate the wing margin enhancer. Chip is essential and also affects expression of a gypsy insertion in Ultrabithorax. Relative to mutations in scalloped or mastermind, a Chip mutation hypersensitizes the wing margin enhancer in cut to gypsy insertions. Therefore, Chip might encode a target of SUHW enhancer-blocking activity.
41
Tabuloc, Christine A., Yao D. Cai, Rosanna S. Kwok, Elizabeth C. Chan, Sergio Hidalgo e Joanna C. Chiu. "CLOCK and TIMELESS regulate rhythmic occupancy of the BRAHMA chromatin-remodeling protein at clock gene promoters". PLOS Genetics 19, n. 2 (21 febbraio 2023): e1010649. http://dx.doi.org/10.1371/journal.pgen.1010649.
Testo completoAbstract (sommario):
Circadian clock and chromatin-remodeling complexes are tightly intertwined systems that regulate rhythmic gene expression. The circadian clock promotes rhythmic expression, timely recruitment, and/or activation of chromatin remodelers, while chromatin remodelers regulate accessibility of clock transcription factors to the DNA to influence expression of clock genes. We previously reported that the BRAHMA (BRM) chromatin-remodeling complex promotes the repression of circadian gene expression in Drosophila. In this study, we investigated the mechanisms by which the circadian clock feeds back to modulate daily BRM activity. Using chromatin immunoprecipitation, we observed rhythmic BRM binding to clock gene promoters despite constitutive BRM protein expression, suggesting that factors other than protein abundance are responsible for rhythmic BRM occupancy at clock-controlled loci. Since we previously reported that BRM interacts with two key clock proteins, CLOCK (CLK) and TIMELESS (TIM), we examined their effect on BRM occupancy to the period (per) promoter. We observed reduced BRM binding to the DNA in clk null flies, suggesting that CLK is involved in enhancing BRM occupancy to initiate transcriptional repression at the conclusion of the activation phase. Additionally, we observed reduced BRM binding to the per promoter in flies overexpressing TIM, suggesting that TIM promotes BRM removal from DNA. These conclusions are further supported by elevated BRM binding to the per promoter in flies subjected to constant light and experiments in Drosophila tissue culture in which the levels of CLK and TIM are manipulated. In summary, this study provides new insights into the reciprocal regulation between the circadian clock and the BRM chromatin-remodeling complex.
42
Xin, Shichao, Jinu Udayabhanu, Xuemei Dai, Yuwei Hua, Yueting Fan, Huasun Huang e Tiandai Huang. "Identification and Functional Evaluation of Three Polyubiquitin Promoters from Hevea brasiliensis". Forests 13, n. 6 (17 giugno 2022): 952. http://dx.doi.org/10.3390/f13060952.
Testo completoAbstract (sommario):
Hevea brasiliensis is an economically important tree species that provides the only commercial source of natural rubber. The replacement of the CaMV35S promoter by endogenous polyubiquitin promoters may be a viable way to improve the genetic transformation of this species. However, no endogenous polyubiquitin promoters in Hevea have been reported yet. Here, we identified three Hevea polyubiquitin genes HbUBI10.1, HbUBI10.2 and HbUBI10.3, which encode ubiquitin monomers having nearly identical amino acid sequences to that of AtUBQ10. The genomic fragments upstream of these HbUBI genes, including the signature leading introns, were amplified as putative HbUBI promoters. In silico analysis showed that a number of cis-acting elements which are conserved within strong constitutive polyubiquitin promoters were presented in these HbUBI promoters. Transcriptomic data revealed that HbUBI10.1 and HbUBI10.2 had a constitutive expression in Hevea plants. Semi-quantitative RT-PCR showed that these three HbUBI genes were expressed higher than the GUS gene driven by CaMV35S in transgenic Hevea leaves. All three HbUBI promoters exhibited the capability to direct GFP expression in both transient and stable transformation assays, although they produced lower protoplast transformation efficiencies than the CaMV35S promoter. These HbUBI promoters will expand the availability of promoters for driving the transgene expression in Hevea genetic engineering.
43
Squire, Derrick J. P., Meng Xu, Jeffrey A. Cole, Stephen J. W. Busby e Douglas F. Browning. "Competition between NarL-dependent activation and Fis-dependent repression controls expression from the Escherichia coli yeaR and ogt promoters". Biochemical Journal 420, n. 2 (13 maggio 2009): 249–57. http://dx.doi.org/10.1042/bj20090183.
Testo completoAbstract (sommario):
The Escherichia coli NarL protein is a global gene regulatory factor that activates transcription at many target promoters in response to nitrate and nitrite ions. Although most NarL-dependent promoters are also co-dependent on a second transcription factor, FNR protein, two targets, the yeaR and ogt promoters, are activated by NarL alone with no involvement of FNR. Biochemical and genetic studies presented here show that activation of the yeaR promoter is dependent on the binding of NarL to a single target centred at position −43.5, whereas activation at the ogt promoter requires NarL binding to tandem DNA targets centred at position −45.5 and −78.5. NarL-dependent activation at both the yeaR and ogt promoters is decreased in rich medium and this depends on Fis, a nucleoid-associated protein. DNase I footprinting studies identified Fis-binding sites that overlap the yeaR promoter NarL site at position −43.5, and the ogt promoter NarL site at position −78.5, and suggest that Fis represses both promoters by displacing NarL. The ogt gene encodes an O6-alkylguanine DNA alkyltransferase and, hence, this is the first report of expression of a DNA repair function being controlled by nitrate ions.
44
Durante-Rodríguez, Gutiérrez-del-Arroyo, Vélez, Díaz e Carmona. "Further Insights into the Architecture of the PN Promoter That Controls the Expression of the bzd Genes in Azoarcus". Genes 10, n. 7 (27 giugno 2019): 489. http://dx.doi.org/10.3390/genes10070489.
Testo completoAbstract (sommario):
The anaerobic degradation of benzoate in bacteria involves the benzoyl-CoA central pathway. Azoarcus/Aromatoleum strains are a major group of anaerobic benzoate degraders, and the transcriptional regulation of the bzd genes was extensively studied in Azoarcus sp. CIB. In this work, we show that the bzdR regulatory gene and the PN promoter can also be identified upstream of the catabolic bzd operon in all benzoate-degrader Azoarcus/Aromatoleum strains whose genome sequences are currently available. All the PN promoters from Azoarcus/Aromatoleum strains described here show a conserved architecture including three operator regions (ORs), i.e., OR1 to OR3, for binding to the BzdR transcriptional repressor. Here, we demonstrate that, whereas OR1 is sufficient for the BzdR-mediated repression of the PN promoter, the presence of OR2 and OR3 is required for de-repression promoted by the benzoyl-CoA inducer molecule. Our results reveal that BzdR binds to the PN promoter in the form of four dimers, two of them binding to OR1. The BzdR/PN complex formed induces a DNA loop that wraps around the BzdR dimers and generates a superstructure that was observed by atomic force microscopy. This work provides further insights into the existence of a conserved BzdR-dependent mechanism to control the expression of the bzd genes in Azoarcus strains.
45
Rollins, Robert A., Patrick Morcillo e Dale Dorsett. "Nipped-B, a Drosophila Homologue of Chromosomal Adherins, Participates in Activation by Remote Enhancers in the cut and Ultrabithorax Genes". Genetics 152, n. 2 (1 giugno 1999): 577–93. http://dx.doi.org/10.1093/genetics/152.2.577.
Testo completoAbstract (sommario):
Abstract How enhancers are able to activate promoters located several kilobases away is unknown. Activation by the wing margin enhancer in the cut gene, located 85 kb from the promoter, requires several genes that participate in the Notch receptor pathway in the wing margin, including scalloped, vestigial, mastermind, Chip, and the Nipped locus. Here we show that Nipped mutations disrupt one or more of four essential complementation groups: l(2)41Ae, l(2)41Af, Nipped-A, and Nipped-B. Heterozygous Nipped mutations modify Notch mutant phenotypes in the wing margin and other tissues, and magnify the effects that mutations in the cis regulatory region of cut have on cut expression. Nipped-A and l(2)41Af mutations further diminish activation by a wing margin enhancer partly impaired by a small deletion. In contrast, Nipped-B mutations do not diminish activation by the impaired enhancer, but increase the inhibitory effect of a gypsy transposon insertion between the enhancer and promoter. Nipped-B mutations also magnify the effect of a gypsy insertion in the Ultrabithorax gene. Gypsy binds the Suppressor of Hairy-wing insulator protein [Su(Hw)] that blocks enhancer-promoter communication. Increased insulation by Su(Hw) in Nipped-B mutants suggests that Nipped-B products structurally facilitate enhancer-promoter communication. Compatible with this idea, Nipped-B protein is homologous to a family of chromosomal adherins with broad roles in sister chromatid cohesion, chromosome condensation, and DNA repair.
46
Sun, Wu-Sheng, Hyeon Yang, Jin Gu No, Haesun Lee, Nahyun Lee, Minguk Lee, Man-Jong Kang e Keon Bong Oh. "Select Porcine Elongation Factor 1α Sequences Mediate Stable High-Level and Upregulated Expression of Heterologous Genes in Porcine Cells in Response to Primate Serum". Genes 12, n. 7 (7 luglio 2021): 1046. http://dx.doi.org/10.3390/genes12071046.
Testo completoAbstract (sommario):
Genetically engineered (GE) pigs with various combinations of genetic profiles have been developed using heterologous promoters. This study aimed to identify autologous promoters for high and ubiquitous expression of xenotransplantation relevant genes in GE pigs. A 1.4 kb upstream regulatory sequence of porcine elongation factor 1α (pEF1α) gene was selected and isolated for use as a promoter. Activity of the pEF1α promoter was subsequently compared with that of the cytomegalovirus (CMV) promoter, CMV enhancer/chicken β-actin (CAG) promoter, and human EF1α (hEF1α) promoter in different types of pig-derived cells. Comparative analysis of luciferase and mutant human leukocyte antigen class E-F2A-β-2 microglobulin (HLA-E) expression driven by pEF1α, CMV, CAG, and hEF1α promoters revealed the pEF1α promoter mediated comparable expression levels with those of the CAG promoter in porcine ear skin fibroblasts (PEFs) and porcine kidney-15 (PK-15) cells, but lower than those of the CAG promoter in porcine aortic endothelial cells (PAECs). The pEF1α promoter provided long-term stable HLA-E expression in PEFs, but the CAG promoter failed to sustain those levels of expression. For xenogeneic serum-induced cytotoxicity assays, the cells were cultured for several hours in growth medium supplemented with primate serum. Notably, the pEF1α promoter induced significant increases in luciferase and HLA-E expression in response to primate serum in PAECs compared with those driven by the CAG promoter, suggesting the pEF1α promoter could regulate temporal expression of heterologous genes under xenogeneic-cytotoxic conditions. These results suggest the pEF1α promoter may be valuable for development of GE pigs spatiotemporally and stably expressing immunomodulatory genes for xenotransplantation.
47
Feng, Xiaofan, e Mario Andrea Marchisio. "Novel S. cerevisiae Hybrid Synthetic Promoters Based on Foreign Core Promoter Sequences". International Journal of Molecular Sciences 22, n. 11 (27 maggio 2021): 5704. http://dx.doi.org/10.3390/ijms22115704.
Testo completoAbstract (sommario):
Promoters are fundamental components of synthetic gene circuits. They are DNA segments where transcription initiation takes place. New constitutive and regulated promoters are constantly engineered in order to meet the requirements for protein and RNA expression into different genetic networks. In this work, we constructed and optimized new synthetic constitutive promoters for the yeast Saccharomyces cerevisiae. We started from foreign (e.g., viral) core promoters as templates. They are, usually, unfunctional in yeast but can be activated by extending them with a short sequence, from the CYC1 promoter, containing various transcription start sites (TSSs). Transcription was modulated by mutating the TATA box composition and varying its distance from the TSS. We found that gene expression is maximized when the TATA box has the form TATAAAA or TATATAA and lies between 30 and 70 nucleotides upstream of the TSS. Core promoters were turned into stronger promoters via the addition of a short UAS. In particular, the 40 nt bipartite UAS from the GPD promoter can enhance protein synthesis considerably when placed 150 nt upstream of the TATA box. Overall, we extended the pool of S. cerevisiae promoters with 59 new samples, the strongest overcoming the native TEF2 promoter.
48
Prelich, G., e F. Winston. "Mutations that suppress the deletion of an upstream activating sequence in yeast: involvement of a protein kinase and histone H3 in repressing transcription in vivo." Genetics 135, n. 3 (1 novembre 1993): 665–76. http://dx.doi.org/10.1093/genetics/135.3.665.
Testo completoAbstract (sommario):
Abstract Regulated transcription of most protein-encoding genes in Saccharomyces cerevisiae requires an upstream activating sequence (UAS); in the absence of UAS elements, little or no transcription occurs. In certain mutant strains, however, promoters that have been deleted for their UAS can direct significant levels of transcription, indicating that the remaining promoter elements (the basal promoter) are capable of directing higher levels of transcription, but they are normally represented in wild-type strains. To analyze this repression, we have selected for mutations that cause increased transcription of the SUC2 gene in the absence of its UAS. In addition to some previously studied genes, this selection has identified five genes that we have designated BUR1, BUR2, BUR3, BUR5 and BUR6 (for Bypass UAS Requirement). The bur mutations cause pleiotropic phenotypes, indicating that they affect transcription of many genes. Furthermore, some bur mutations suppress the requirement for the SNF5 trans-activator at both SUC2 and Ty. Additional analysis has demonstrated that BUR1 is identical to SGV1, which encodes a CDC28-related protein kinase. This result indicates that protein phosphorylation is important for repression of the SUC2 basal promoter as well as other aspects of transcription in vivo. Finally, BUR5 is identical to HHT1, encoding histone H3, further implicating chromatin structure as important for expression of SUC2.
49
Kanno, Alex I., Cibelly Goulart, Henrique K. Rofatto, Sergio C. Oliveira, Luciana C. C. Leite e Johnjoe McFadden. "New Recombinant Mycobacterium bovis BCG Expression Vectors: Improving Genetic Control over Mycobacterial Promoters". Applied and Environmental Microbiology 82, n. 8 (5 febbraio 2016): 2240–46. http://dx.doi.org/10.1128/aem.03677-15.
Testo completoAbstract (sommario):
ABSTRACTThe expression of many antigens, stimulatory molecules, or even metabolic pathways in mycobacteria such asMycobacterium bovisBCG orM. smegmatiswas made possible through the development of shuttle vectors, and several recombinant vaccines have been constructed. However, gene expression in any of these systems relied mostly on the selection of natural promoters expected to provide the required level of expression by trial and error. To establish a systematic selection of promoters with a range of strengths, we generated a library of mutagenized promoters through error-prone PCR of the strong PL5promoter, originally from mycobacteriophage L5. These promoters were cloned upstream of the enhanced green fluorescent protein reporter gene, and recombinantM. smegmatisbacteria exhibiting a wide range of fluorescence levels were identified. A set of promoters was selected and identified as having high (pJK-F8), intermediate (pJK-B7, pJK-E6, pJK-D6), or low (pJK-C1) promoter strengths in bothM. smegmatisandM. bovisBCG. The sequencing of the promoter region demonstrated that it was extensively modified (6 to 11%) in all of the plasmids selected. To test the functionality of the system, two different expression vectors were demonstrated to allow corresponding expression levels of theSchistosoma mansoniantigen Sm29 in BCG. The approach used here can be used to adjust expression levels for synthetic and/or systems biology studies or for vaccine development to maximize the immune response.
50
Freedman, Jennifer A., e Sue Jinks-Robertson. "Genetic Requirements for Spontaneous and Transcription-Stimulated Mitotic Recombination inSaccharomyces cerevisiae". Genetics 162, n. 1 (1 settembre 2002): 15–27. http://dx.doi.org/10.1093/genetics/162.1.15.
Testo completoAbstract (sommario):
AbstractThe genetic requirements for spontaneous and transcription-stimulated mitotic recombination were determined using a recombination system that employs heterochromosomal lys2 substrates that can recombine only by crossover or only by gene conversion. The substrates were fused either to a constitutive low-level promoter (pLYS) or to a highly inducible promoter (pGAL). In the case of the “conversion-only” substrates the use of heterologous promoters allowed either the donor or the recipient allele to be highly transcribed. Transcription of the donor allele stimulated gene conversions in rad50, rad51, rad54, and rad59 mutants, but not in rad52, rad55, and rad57 mutants. In contrast, transcription of the recipient allele stimulated gene conversions in rad50, rad51, rad54, rad55, rad57, and rad59 mutants, but not in rad52 mutants. Finally, transcription stimulated crossovers in rad50, rad54, and rad59 mutants, but not in rad51, rad52, rad55, and rad57 mutants. These data are considered in relation to previously proposed molecular mechanisms of transcription-stimulated recombination and in relation to the roles of the recombination proteins.