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https://www.readbyqxmd.com/read/28324615/construction-of-low-ethanol-wine-yeasts-through-partial-deletion-of-the-saccharomyces-cerevisiae-pdc2-gene
#1
Raúl Andrés Cuello, Karina Johana Flores Montero, Laura Analía Mercado, Mariana Combina, Iván Francisco Ciklic
We propose an alternative GMO based strategy to obtain Saccharomyces cerevisiae mutant strains with a slight reduction in their ability to produce ethanol, but with a moderate impact on the yeast metabolism. Through homologous recombination, two truncated Pdc2p proteins Pdc2pΔ344 and Pdc2pΔ519 were obtained and transformed into haploid and diploid lab yeast strains. In the pdc2Δ344 mutants the DNA-binding and transactivation site of the protein remain intact, whereas in pdc2Δ519 only the DNA-binding site is conserved...
December 2017: AMB Express
https://www.readbyqxmd.com/read/28324508/in-vitro-preparation-and-crystallization-of-vertebrate-telomerase-subunits
#2
Jing Huang, Christopher J Bley, Dustin P Rand, Julian J L Chen, Ming Lei
Telomerase is a unique reverse transcriptase that replicates the telomeric DNA at most eukaryotic chromosomal ends. The telomerase consists of the catalytic protein subunit TERT and the RNA component TR that provides the template for telomeric DNA synthesis. In vitro reconstitution of telomerase core components in large quantity is the prerequisite to studying the catalytic mechanisms of telomerase at the structural level; however, large-scale preparation of recombinant telomerase, especially that of higher eukaryotes, has been a big challenge for a long time...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28324143/rapid-customised-operon-assembly-by-yeast-recombinational-cloning
#3
Michael A Liu, Johanna J Kenyon, Jason Lee, Peter R Reeves
We have developed a system called the Operon Assembly Protocol (OAP), which takes advantage of the homologous recombination DNA repair pathway in Saccharomyces cerevisiae to assemble full-length operons from a series of overlapping PCR products into a specially engineered yeast-Escherichia coli shuttle vector. This flexible, streamlined system can be used to assemble several operon clones simultaneously, and each clone can be expressed in the same E. coli tester strain to facilitate direct functional comparisons...
March 21, 2017: Applied Microbiology and Biotechnology
https://www.readbyqxmd.com/read/28319081/guide-independent-dna-cleavage-by-archaeal-argonaute-from-methanocaldococcus-jannaschii
#4
Adrian Zander, Sarah Willkomm, Sapir Ofer, Marleen van Wolferen, Luisa Egert, Sabine Buchmeier, Sarah Stöckl, Philip Tinnefeld, Sabine Schneider, Andreas Klingl, Sonja-Verena Albers, Finn Werner, Dina Grohmann
Prokaryotic Argonaute proteins acquire guide strands derived from invading or mobile genetic elements, via an unknown pathway, to direct guide-dependent cleavage of foreign DNA. Here, we report that Argonaute from the archaeal organism Methanocaldococcus jannaschii (MjAgo) possesses two modes of action: the canonical guide-dependent endonuclease activity and a non-guided DNA endonuclease activity. The latter allows MjAgo to process long double-stranded DNAs, including circular plasmid DNAs and genomic DNAs...
March 20, 2017: Nature Microbiology
https://www.readbyqxmd.com/read/28319063/compromised-brca1-palb2-interaction-is-associated-with-breast-cancer-risk
#5
T K Foo, M Tischkowitz, S Simhadri, T Boshari, N Zayed, K A Burke, S H Berman, P Blecua, N Riaz, Y Huo, Y C Ding, S L Neuhausen, B Weigelt, J S Reis-Filho, W D Foulkes, B Xia
The major breast cancer suppressor proteins BRCA1 and BRCA2 play essential roles in homologous recombination (HR)-mediated DNA repair, which is thought to be critical for tumor suppression. The two BRCA proteins are linked by a third tumor suppressor, PALB2, in the HR pathway. While truncating mutations in these genes are generally pathogenic, interpretation of missense variants remains a challenge. To date, patient-derived missense variants that disrupt PALB2 binding have been identified in BRCA1 and BRCA2; however, there has not been sufficient evidence to prove their pathogenicity in humans, and no variants in PALB2 that disrupt either its BRCA1 or BRCA2 binding have been reported...
March 20, 2017: Oncogene
https://www.readbyqxmd.com/read/28318385/arsenic-induced-sumoylation-of-mus81-is-involved-in-regulating-genomic-stability
#6
Liyan Hu, Feikun Yang, Lou Lu, Wei Dai
Chronic environmental exposure to metal toxicants such as chromium and arsenic is closely related to the development of several types of common cancers. Genetic and epigenetic studies in the past decade reveal that post-translational modifications of histones play a role in metal carcinogenesis. However, exact molecular mechanisms of metal carcinogenesis remain to be elucidated. In this study we found that As2O3, an environmental metal toxicant, up-regulated overall modifications of many cellular proteins by SUMO2/3...
March 20, 2017: Cell Cycle
https://www.readbyqxmd.com/read/28317934/dek-is-required-for-homologous-recombination-repair-of-dna-breaks
#7
Eric A Smith, Boris Gole, Nicholas A Willis, Rebeca Soria, Linda M Starnes, Eric F Krumpelbeck, Anil G Jegga, Abdullah M Ali, Haihong Guo, Amom R Meetei, Paul R Andreassen, Ferdinand Kappes, Lisa M Privette Vinnedge, Jeremy A Daniel, Ralph Scully, Lisa Wiesmüller, Susanne I Wells
DEK is a highly conserved chromatin-bound protein whose upregulation across cancer types correlates with genotoxic therapy resistance. Loss of DEK induces genome instability and sensitizes cells to DNA double strand breaks (DSBs), suggesting defects in DNA repair. While these DEK-deficiency phenotypes were thought to arise from a moderate attenuation of non-homologous end joining (NHEJ) repair, the role of DEK in DNA repair remains incompletely understood. We present new evidence demonstrating the observed decrease in NHEJ is insufficient to impact immunoglobulin class switching in DEK knockout mice...
March 20, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28317025/marker-recycling-in-candida-albicans-through-crispr-cas9-induced-marker-excision
#8
Manning Y Huang, Aaron P Mitchell
We describe here a new approach to marker recycling, a controlled sequence of steps in which a genetic marker is selected and then lost. Marker recycling is important for genetic manipulation, because it allows a single selection marker to be used repeatedly. Our approach relies upon the ability of the CRISPR-Cas9 system to make a targeted double-strand break in DNA and the expectation that a double-strand break within a selection marker may promote recombination between directly repeated sequences that flank the marker...
March 2017: MSphere
https://www.readbyqxmd.com/read/28316093/morphology-heterogeneity-within-a-campylobacter-jejuni-helical-population-the-use-of-calcofluor-white-to-generate-rod-shaped-c-jejuni-81-176-clones-and-the-genetic-determinants-responsible-for-differences-in-morphology-within-11168-strains
#9
Emilisa Frirdich, Jacob Biboy, Steven Huynh, Craig T Parker, Waldemar Vollmer, Erin C Gaynor
Campylobacter jejuni helical shape is important for colonization and host interactions with straight mutants having altered biological properties. Passage on calcofluor white (CFW) resulted in C. jejuni 81-176 isolates with morphology changes: either a straight morphology from frameshift mutations and single nucleotide polymorphisms in peptidoglycan hydrolase genes pgp1 or pgp2 or a reduction in curvature due a frameshift mutation in cjj81176_1105, a putative peptidoglycan endopeptidase. Shape defects were restored by complementation...
March 18, 2017: Molecular Microbiology
https://www.readbyqxmd.com/read/28316014/rescue-of-recombinant-newcastle-disease-virus-a-short-history-of-how-it-all-started
#10
REVIEW
Aidin Molouki, Ben Peeters
Reverse genetics of viruses has come a long way, and many recombinant viruses have been generated since the first successful "rescues" were reported in the late 1970s. Recombinant Newcastle disease virus (rNDV), a non-segmented negative-sense RNA virus (NSNSV), was first rescued in 1999 using a reverse genetics approach similar to that reported for other recombinant viruses of the order Mononegavirales a few years before. The route from an original NDV isolate to the generation of its recombinant counterpart requires many steps that have to be sequentially and carefully completed...
March 18, 2017: Archives of Virology
https://www.readbyqxmd.com/read/28315926/dissection-of-a-major-qtl-qhir1-conferring-maternal-haploid-induction-ability-in-maize
#11
Sudha K Nair, Willem Molenaar, Albrecht E Melchinger, Prasanna M Boddupalli, Leocadio Martinez, Luis Antonio Lopez, Vijay Chaikam
Among the qhir11 and qhir12 sub-regions of a major QTL qhir1, only qhir11 has significant effect on maternal haploid induction, segregation distortion and kernel abortion. In vivo haploid induction in maize can be triggered in high frequencies by pollination with special genetic stocks called haploid inducers. Several genetic studies with segregating populations from non-inducer x inducer crosses identified a major QTL, qhir1, on chromosome 1.04 contributing to in vivo haploid induction. A recent Genome Wide Association Study using 51 inducers and 1482 non-inducers also identified two sub-regions within the qhir1 QTL region, named qhir11 and qhir12; qhir12 was proposed to be mandatory for haploid induction because the haplotype of qhir11 was also present in some non-inducers and putative candidate genes coding for DNA and amino acid binding proteins were identified in the qhir12 region...
March 18, 2017: TAG. Theoretical and Applied Genetics. Theoretische und Angewandte Genetik
https://www.readbyqxmd.com/read/28315830/paternal-induction-of-hybrid-dysgenesis-in-drosophila-melanogaster-is-weakly-correlated-with-both-p-element-and-hobo-element-dosage
#12
Satyam P Srivastav, Erin S Kelleher
Transposable elements (TEs) are virtually ubiquitous components of genomes, yet they often impose significant fitness consequences on their hosts. In addition to producing specific deleterious mutations by insertional inactivation, TEs also impose general fitness costs by inducing DNA damage and participating in ectopic recombination. These latter fitness costs are often assumed to be dosage-dependent, with stronger effects occurring in the presence of higher TE copy numbers. We test this assumption in Drosophila melanogaster by considering the relationship between the copy number of two active DNA transposons, P-element and hobo element, and the incidence of hybrid dysgenesis, a sterility syndrome associated with transposon activity in the germline...
March 17, 2017: G3: Genes—Genomes—Genetics
https://www.readbyqxmd.com/read/28315507/polymorphisms-and-mutations-in-gstp1-rad51-xrcc1-and-xrcc3-genes-in-breast-cancer-patients
#13
Mazhar Salim Al Zoubi, Katia Zavaglia, Chiara Mazanti, Mohammad Al Hamad, Khalid Al Batayneh, Alaa A A Aljabali, Generoso Bevilacqua
BACKGROUND: Genotoxic factors, including ionizing radiation and oxidative stress, are associated with genomic instability and development of breast cancer (BC). The homologous recombination DNA repair (HRR) pathway, base excision repair (BER) mechanism, and antioxidative enzymes are required as defense mechanisms against these DNA damaging agents. GSTP1, XRCC1, XRCC3 and RAD51 proteins are essential components of antioxidation, BER and HRR of DNA, respectively. Deficiencies in BER, HRR and antioxidation pathways are involved in the progression of cancer...
March 6, 2017: International Journal of Biological Markers
https://www.readbyqxmd.com/read/28315209/recombinant-allergen-production-in-e-coli
#14
Changqi Liu, LeAnna N Willison, Shridhar K Sathe
Recombinant protein allergens have been used in allergy studies, allergy diagnosis, and epitope mapping. Messenger RNAs (mRNAs) are isolated from tissues of interest for complementary DNA (cDNA) library construction. Subsequently, the allergen gene is amplified by polymerase chain reaction (PCR) and sequenced. The amplified gene is then cloned into an expression vector, expressed in Escherichia coli cells, and purified from the cell lysate. This chapter describes the protocols for recombinant allergen production...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28314872/rt-qpcr-with-chimeric-du-stem-loop-primer-is-efficient-for-the-detection-of-bacterial-small-rnas
#15
Yangfan Wu, Xuejiao Xing, Ting You, Rubing Liang, Jianhua Liu
Small non-coding RNAs are considered be involved in the regulation of multiple cellular processes. Quantitative reverse transcription PCR (RT-qPCR) is widely used in the detection of eukaryotic microRNA, and the stem-loop primers can improve the specificity and efficiency of reverse transcription. However, the loop structure of primers probably influence the next quantitative amplification due to the base stacking and steric hindrance. Here, we designed a chimeric stem-loop primer with a deoxyuracil (dU) base located near the RNA matching part...
March 17, 2017: Applied Microbiology and Biotechnology
https://www.readbyqxmd.com/read/28314074/better-understanding-of-homologous-recombination-through-a-12-week-laboratory-course-for-undergraduates-majoring-in-biotechnology
#16
Ming Li, Xiaodong Shen, Yan Zhao, Xiaomei Hu, Fuquan Hu, Xiancai Rao
Homologous recombination, a central concept in biology, is defined as the exchange of DNA strands between two similar or identical nucleotide sequences. Unfortunately, undergraduate students majoring in biotechnology often experience difficulties in understanding the molecular basis of homologous recombination. In this study, we developed and implemented a 12-week laboratory course for biotechnology undergraduates in which gene targeting in Streptococcus suis was used to facilitate their understanding of the basic concept and process of homologous recombination...
March 17, 2017: Biochemistry and Molecular Biology Education
https://www.readbyqxmd.com/read/28303813/molecular-typing-of-chlamydia-trachomatis-an-overview
#17
REVIEW
Jyoti Rawre, Deepak Juyal, Benu Dhawan
Urogenital infection due to Chlamydia trachomatis (CT) is one of the most common bacterial sexually transmitted infections (STIs) and is a major public health problem worldwide. Molecular characterisation of CT is important for understanding the pathophysiological mechanisms of chlamydial disease and its transmission dynamics in sexual networks. Traditionally, strain typing of CT was based on serotyping methods characterising the major outer membrane protein (MOMP). With the advent of polymerase chain reaction and sequencing the era of molecular typing began...
January 2017: Indian Journal of Medical Microbiology
https://www.readbyqxmd.com/read/28303365/immunoglobulin-like-domain-4-mediated-ligand-independent-dimerization-triggers-vegfr-2-activation-in-huvecs-and-vegfr2-positive-breast-cancer-cells
#18
Sheng Zhang, Xiaoping Gao, Wei Fu, Shengwei Li, Limin Yue
PURPOSE: The extracellular region (EC) of the vascular endothelial growth factor (VEGF) receptor-2 (VEGFR-2) contains seven immunoglobulin-like (Ig-like) domains that are required for specific ligand binding and receptor dimerization. Studies of domain 4-7 deletions and substitutions provided insights into the interaction between receptors in the absence of VEGF. In this study, we investigated the effect of domain 4 in ligand-independent VEGFR-2 dimerization and activation in human vascular endothelial cells and human breast cancer cells...
March 16, 2017: Breast Cancer Research and Treatment
https://www.readbyqxmd.com/read/28301581/assessing-tn5-and-sleeping-beauty-for-transpositional-transgenesis-by-cytoplasmic-injection-into-bovine-and-ovine-zygotes
#19
R J Bevacqua, R Fernandez-Martin, N G Canel, A Gibbons, D Texeira, F Lange, G Vans Landschoot, V Savy, O Briski, M I Hiriart, E Grueso, Z Ivics, O Taboga, W A Kues, S Ferraris, D F Salamone
Transgenic domestic animals represent an alternative to bioreactors for large-scale production of biopharmaceuticals and could also provide more accurate biomedical models than rodents. However, their generation remains inefficient. Recently, DNA transposons allowed improved transgenesis efficiencies in mice and pigs. In this work, Tn5 and Sleeping Beauty (SB) transposon systems were evaluated for transgenesis by simple cytoplasmic injection in livestock zygotes. In the case of Tn5, the transposome complex of transposon nucleic acid and Tn5 protein was injected...
2017: PloS One
https://www.readbyqxmd.com/read/28300166/oepr-cloning-an-efficient-and-seamless-cloning-strategy-for-large-and-multi-fragments
#20
Chang-Jun Liu, Hui Jiang, Lei Wu, Ling-Yun Zhu, Er Meng, Dong-Yi Zhang
Here, an efficient cloning strategy for large DNA fragments and for simultaneous assembly of multiple DNA fragments assembly is presented. This strategy is named OEPR (based on Overlap Extension PCR and Recombination in vivo). OEPR cloning is a seamless, restriction- and ligation-independent method. The method takes advantage of both homologous recombination enzymes in E. coli and overlap PCR. Using OEPR cloning, a long fragment (1-6 kb) or multiple fragments (2-4 fragments) can be easily constructed and simultaneously assembled into a target vector...
March 16, 2017: Scientific Reports
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