Insertion-Duplication Mutagenesis inStreptococcus pneumoniae: Targeting Fragment Length Is a Critical Parameter in Use as a Random Insertion Tool

1998 ◽  
Vol 64 (12) ◽  
pp. 4796-4802 ◽  
Author(s):  
Myeong S. Lee ◽  
Chaok Seok ◽  
Donald A. Morrison
Keyword(s):  
Gene Knockout ◽  
Genomic Analysis ◽  
Combinatorial Theory ◽  
Donor Plasmid ◽  
Uniform Size ◽  
Knock Out ◽  
Duplication Events ◽  
Simplifying Assumptions ◽  
Insertion Tool ◽  
Random Insertion

ABSTRACT To examine whether insertion-duplication mutagenesis with chimeric DNA as a transformation donor could be valuable as a gene knockout tool for genomic analysis in Streptococcus pneumoniae, we studied the transformation efficiency and targeting specificity of the process by using a nonreplicative vector with homologous targeting inserts of various sizes. Insertional recombination was very specific in targeting homologous sites. While the recombination rate did not depend on which site or region was targeted, it did depend strongly on the size of the targeting insert in the donor plasmid, in proportion to the fifth power of its length for inserts of 100 to 500 bp. The dependence of insertion-duplication events on the length of the targeting homology was quite different from that for linear allele replacement and places certain limits on the design of mutagenesis experiments. The number of independent pneumococcal targeting fragments of uniform size required to knock out any desired fraction of the genes in a model genome with a defined probability was calculated from these data by using a combinatorial theory with simplifying assumptions. The results show that efficient and thorough mutagenesis of a large part of the pneumococcal genome should be practical when using insertion-duplication mutagenesis.

Comparative genomic analysis of a mammalian β-defensin gene cluster

2007 ◽  
Vol 30 (3) ◽  
pp. 213-222 ◽  
Author(s):  
Yashwanth Radhakrishnan ◽  
Mario A. Fares ◽  
Frank S. French ◽  
Susan H. Hall
Keyword(s):  
Positive Selection ◽  
Gene Cluster ◽  
Genomic Analysis ◽  
Urogenital Tract ◽  
Comparative Genomic ◽  
Evolutionary Analysis ◽  
Defensin Gene ◽  
Duplication Events ◽  
Molecular Evolutionary Analysis

Comparative genomic analyses have yielded valuable insights into conserved and divergent aspects of gene function, regulation, and evolution. Herein, we describe the characterization of a mouse β-defensin gene cluster locus on chromosome 2F6. In addition, we present the evolutionary analysis of this cluster and its human, rhesus, and rat orthologs. Expression analysis in mouse revealed the occurrence of defensin cluster transcripts in multiple tissues, with the highest abundance in the urogenital tract. Molecular evolutionary analysis suggests that this cluster originated by a series of duplication events, and by positive selection occurring even after the rodent-primate split. In addition, the constraints analysis showed higher positive selection in rodents than in primates, especially distal to the six-cysteine array. Positive selection in the evolution of these defensins may relate not only to the evolving enhancement of ancestral host defense but also to functional innovations in reproduction. The multiplicity of defensins and their preferential overexpression in the urogenital tract indicate that defensins function in the protection and maintenance of fertility.

scholarly journals In silico gene knockout prediction using a hybrid of Bat algorithm and minimization of metabolic adjustment

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Mei Yen Man ◽  
Mohd Saberi Mohamad ◽  
Yee Wen Choon ◽  
Mohd Arfian Ismail
Keyword(s):  
Metabolic Engineering ◽  
Gene Knockout ◽  
Lactate Production ◽  
Bat Algorithm ◽  
Host Cells ◽  
Knock Out ◽  
Genetic Manipulations ◽  
Microbial Strains ◽  
Metabolic Adjustment ◽  
Minimization Of Metabolic Adjustment

Abstract Microorganisms commonly produce many high-demand industrial products like fuels, food, vitamins, and other chemicals. Microbial strains are the strains of microorganisms, which can be optimized to improve their technological properties through metabolic engineering. Metabolic engineering is the process of overcoming cellular regulation in order to achieve a desired product or to generate a new product that the host cells do not usually need to produce. The prediction of genetic manipulations such as gene knockout is part of metabolic engineering. Gene knockout can be used to optimize the microbial strains, such as to maximize the production rate of chemicals of interest. Metabolic and genetic engineering is important in producing the chemicals of interest as, without them, the product yields of many microorganisms are normally low. As a result, the aim of this paper is to propose a combination of the Bat algorithm and the minimization of metabolic adjustment (BATMOMA) to predict which genes to knock out in order to increase the succinate and lactate production rates in Escherichia coli (E. coli).

scholarly journals Gene Duplication and Gain in the Trematode Atriophallophorus Winterbourni Contributes to Adaptation to Parasitism

2021 ◽  
Author(s):  
Natalia Zajac ◽  
Stefan Zoller ◽  
Katri Seppälä ◽  
David Moi ◽  
Christophe Dessimoz ◽  
...  
Keyword(s):  
De Novo ◽  
Genomic Analysis ◽  
Enrichment Analysis ◽  
Gene Families ◽  
Comparative Genomic ◽  
Genomic Changes ◽  
Go Enrichment ◽  
Duplication Events ◽  
Parasitic Lifestyle ◽  
Host Parasite

Abstract Gene duplications and novel genes have been shown to play a major role in helminth adaptation to a parasitic lifestyle because they provide the novelty necessary for adaptation to a changing environment, such as living in multiple hosts. Here we present the de novo sequenced and annotated genome of the parasitic trematode Atriophallophorus winterbourni and its comparative genomic analysis to other major parasitic trematodes. First, we reconstructed the species phylogeny, and dated the split of A. winterbourni from the Opisthorchiata suborder to approximately 237.4 MYA (± 120.4 MY). We then addressed the question of which expanded gene families and gained genes are potentially involved in adaptation to parasitism. To do this, we used Hierarchical Orthologous Groups to reconstruct three ancestral genomes on the phylogeny leading to A. winterbourni and performed a GO enrichment analysis of the gene composition of each ancestral genome, allowing us to characterize the subsequent genomic changes. Out of the 11,499 genes in the A. winterbourni genome, as much as 24% have arisen through duplication events since the speciation of A. winterbourni from the Opisthorchiata, and as much as 31.9% appear to be novel, i.e. newly acquired. We found 13 gene families in A. winterbourni to have had more than 10 genes arising through these recent duplications; all of which have functions potentially relating to host behavioural manipulation, host tissue penetration, and hiding from host immunity through antigen presentation. We identified several families with genes evolving under positive selection. Our results provide a valuable resource for future studies on the genomic basis of adaptation to parasitism and point to specific candidate genes putatively involved in antagonistic host-parasite adaptation.

scholarly journals TMOD-04. CHARACTERIZATION OF MUTANT IDH1 OLIGODENDROGLIOMAS

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi263-vi263
Author(s):  
Rick Heinz ◽  
Sheri Holmen
Keyword(s):  
Molecular Mechanisms ◽  
Gene Knockout ◽  
Genomic Analysis ◽  
World Health ◽  
Low Grade ◽  
Idh Mutation ◽  
Loss Of Function ◽  
In Vivo Models ◽  
Mutant Idh1

Abstract Gliomas are the most common primary central nervous system malignancy in adults, but the molecular mechanisms responsible for their development and progression are not fully understood. Recent genomic analysis of World Health Organization grade II-III gliomas identifies 3 molecular subtypes of low grade glioma: no IDH mutation; IDH mutation without 1p/19q co-deletion; and IDH mutation with 1p/19q co-deletion. The latter, categorized as oligodendroglioma, commonly expresses loss of function mutations in CIC, FUBP1, and activation of PIK3CA. However, it is unknown if any of these mutations are sufficient to promote glioma development in cooperation with mutant IDH. Furthermore, research in oligodendroglioma is hampered by the lack of in vivo models of this specific glioma subtype. By utilizing the established RCAS/TVA somatic cell gene delivery method, mutant IDH1 can be expressed in the brains of mice. The tumorgenicity of other mutated genes associated with oligodendroglioma can be determined using additional methods such as conditional gene knockout and in vivo CRISPR-Cas9 mediated deletion. A mouse model for oligodendroglioma may identify new targetable genetic drivers of oligodendroglioma that will be useful for testing novel therapeutic strategies.

scholarly journals Genome-wide CRISPR-based gene knockout screens reveal cellular factors and pathways essential for nasopharyngeal carcinoma

2019 ◽  
Vol 294 (25) ◽  
pp. 9734-9745 ◽  
Author(s):  
Chong Wang ◽  
Sizun Jiang ◽  
Liangru Ke ◽  
Luyao Zhang ◽  
Difei Li ◽  
...  
Keyword(s):  
Nasopharyngeal Carcinoma ◽  
Advanced Disease ◽  
Gene Knockout ◽  
Histone Acetyl Transferase ◽  
Knock Out ◽  
Genome Wide ◽  
A Genome ◽  
Cellular Factors ◽  
New Treatments ◽  
Specific Symptoms

Early diagnosis of nasopharyngeal carcinoma (NPC) is difficult because of a lack of specific symptoms. Many patients have advanced disease at diagnosis, and these patients respond poorly to treatment. New treatments are therefore needed to improve the outcome of NPC. To better understand the molecular pathogenesis of NPC, here we used an NPC cell line in a genome-wide CRISPR-based knockout screen to identify the cellular factors and pathways essential for NPC (i.e. dependence factors). This screen identified the Moz, Ybf2/Sas3, Sas2, Tip60 histone acetyl transferase complex, NF-κB signaling, purine synthesis, and linear ubiquitination pathways; and MDM2 proto-oncogene as NPC dependence factors/pathways. Using gene knock out, complementary DNA rescue, and inhibitor assays, we found that perturbation of these pathways greatly reduces the growth of NPC cell lines but does not affect growth of SV40-immortalized normal nasopharyngeal epithelial cells. These results suggest that targeting these pathways/proteins may hold promise for achieving better treatment of patients with NPC.

scholarly journals CRISPR/Cas9-Mediated SlMYBS2 Mutagenesis Reduces Tomato Resistance to Phytophthora infestans

2021 ◽  
Vol 22 (21) ◽  
pp. 11423
Author(s):  
Chunxin Liu ◽  
Yiyao Zhang ◽  
Yinxiao Tan ◽  
Tingting Zhao ◽  
Xiangyang Xu ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Late Blight ◽  
Phytophthora Infestans ◽  
Gene Knockout ◽  
Disease Index ◽  
Oxygen Species ◽  
Wild Type ◽  
Pr Genes ◽  
Knock Out ◽  
Pathogenesis Related

Phytophthora infestans (P. infestans) recently caused epidemics of tomato late blight. Our study aimed to identify the function of the SlMYBS2 gene in response to tomato late blight. To further investigate the function of SlMYBS2 in tomato resistance to P. infestans, we studied the effects of SlMYBS2 gene knock out. The SlMYBS2 gene was knocked out by CRISPR-Cas9, and the resulting plants (SlMYBS2 gene knockout, slmybs2-c) showed reduced resistance to P. infestans, accompanied by increases in the number of necrotic cells, lesion sizes, and disease index. Furthermore, after P. infestans infection, the expression levels of pathogenesis-related (PR) genes in slmybs2-c plants were significantly lower than those in wild-type (AC) plants, while the number of necrotic cells and the accumulation of reactive oxygen species (ROS) were higher than those in wild-type plants. Taken together, these results indicate that SlMYBS2 acts as a positive regulator of tomato resistance to P. infestans infection by regulating the ROS level and the expression level of PR genes.

scholarly journals The Functional Association of ACQOS/VICTR with Salt Stress Resistance in Arabidopsis thaliana Was Confirmed by CRISPR-Mediated Mutagenesis

2021 ◽  
Vol 22 (21) ◽  
pp. 11389
Author(s):  
Sang-Tae Kim ◽  
Minkyung Choi ◽  
Su-Ji Bae ◽  
Jin-Soo Kim
Keyword(s):  
Salt Stress ◽  
Target Genes ◽  
Gene Knockout ◽  
Salt Resistance ◽  
Knock Out ◽  
Chlorophyll Contents ◽  
Guide Rnas ◽  
Osmotic Tolerance ◽  
Multiple Genes ◽  
Acclimation Period

Clustered regularly interspaced palindromic repeat (CRISPR)-mediated mutagenesis has become an important tool in plant research, enabling the characterization of genes via gene knock-out. CRISPR genome editing tools can be applied to generate multi-gene knockout lines. Typically, multiple single-stranded, single guide RNAs (gRNAs) must be expressed in an organism to target multiple genes simultaneously; however, a single gRNA can target multiple genes if the target genes share similar sequences. A gene cluster comprising ACQUIRED OSMOTOLERANCE (ACQOS; AT5G46520) and neighboring nucleotide-binding leucine-rich repeats (NLRs; AT5G46510) is associated with osmotic tolerance. To investigate the role of ACQOS and the tandemly arranged NLR in osmotic tolerance, we introduced small insertion/deletion mutations into two target genes using a single gRNA and obtained transformant plant lines with three different combinations of mutant alleles. We then tested our mutant lines for osmotic tolerance after a salt-stress acclimation period by determining the chlorophyll contents of the mutant seedlings. Our results strongly suggest that ACQOS is directly associated with salt resistance, while the neighboring NLR is not. Here, we confirmed previous findings suggesting the involvement of ACQOS in salt tolerance and demonstrated the usefulness of CRISPR-mediated mutagenesis in validating the functions of genes in a single genetic background.

scholarly journals Noninvasive sleep monitoring in large-scale screening of knock-out mice reveals novel sleep-related genes

2019 ◽  
Author(s):  
Shreyas S. Joshi ◽  
Mansi Sethi ◽  
Martin Striz ◽  
Neil Cole ◽  
James M. Denegre ◽  
...  
Keyword(s):  
Knockout Mice ◽  
Large Scale ◽  
Gene Knockout ◽  
Single Gene ◽  
Mammalian Species ◽  
Sleep Behavior ◽  
Knock Out ◽  
Circadian Phase ◽  
Gene Knockout Mice ◽  
Normal Sleep

AbstractSleep is a critical process that is well-conserved across mammalian species, and perhaps most animals, yet its functions and underlying mechanisms remain poorly understood. Identification of genes and pathways that can influence sleep may shed new light on these functions. Genomic screens enable the detection of previously unsuspected molecular processes that influence sleep. In this study, we report results from a large-scale phenotyping study of sleep-wake parameters for a population of single-gene knockout mice. Sleep-wake parameters were measured using a high throughput, non-invasive piezoelectric system called PiezoSleep. Knockout mice generated on a C57BL6/N (B6N) background were monitored for sleep and wake parameters for five days. By analyzing data from over 6000 mice representing 343 single gene knockout lines, we identified 122 genes influencing traits like sleep duration and bout length that have not been previously implicated in sleep, including those that affect sleep only during a specific circadian phase. PiezoSleep also allows assessment of breath rates during sleep and this was integrated as a supplemental tool in identifying aberrant physiology in these knockout lines. Sex differences were evident in both normal and altered sleep behavior. Through a combination of genetic and phenotypic associations, and known QTLs for sleep, we propose a set of candidate genes playing specific roles in sleep. The high “hit rate” demonstrates that many genes can alter normal sleep behaviors through a variety of mechanisms. Further investigation of these genes may provide insight into the pathways regulating sleep, functional aspects of sleep, or indirect potentially pathological processes that alter normal sleep.

scholarly journals An Improved Method to Knock Out theasdGene ofSalmonella entericaSerovar Pullorum

2009 ◽  
Vol 2009 ◽  
pp. 1-8 ◽  
Author(s):  
Shi-Zhong Geng ◽  
Xin-An Jiao ◽  
Zhi-Ming Pan ◽  
Xiao-Juan Chen ◽  
Xiao-Ming Zhang ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Gene Knockout ◽  
Vector System ◽  
Improved Method ◽  
Suicide Plasmid ◽  
Knock Out ◽  
Suicide Vector ◽  
Conventional Methods

Anasd-deleted (Δasd) mutant ofSalmonella entericaserovar Pullorum (SP) was constructed using an improved method of gene knockout by combining theπ-suicide plasmid system with the Red Disruption system. Theasdgene was efficiently knocked out by the recombinant suicide vector, which replaced theasdgene with theCmRgene. Based on the balanced lethal host-vector system, the phenotype of theΔasdmutant was further defined. The improved method was simpler and more effective than previously reported conventional methods.

scholarly journals Genomic analysis based on chromosome-level genome assembly reveals an expansion of terpene biosynthesis of Azadirachta indica

2021 ◽  
Author(s):  
Yuhui Du ◽  
Wei Song ◽  
Zhiqiu Yin ◽  
shengbo wu ◽  
jiaheng liu ◽  
...  
Keyword(s):  
Azadirachta Indica ◽  
Genome Assembly ◽  
Genomic Analysis ◽  
Comparative Genomic Analysis ◽  
Genomic Research ◽  
Comparative Genomic ◽  
Terpene Biosynthesis ◽  
Chromosome 13 ◽  
Duplication Events ◽  
Chromosome Level

Azadirachta indica (neem), an evergreen tree of the Meliaceae family, is a source of the potent biopesticide azadirachtin. The lack of a chromosome-level assembly impedes the understanding of in-depth genomic architecture and the comparative genomic analysis of A. indica. Here, a high-quality genome assembly of A. indica was constructed using a combination of data from Illumina, PacBio, and Hi-C technology, which is the first chromosome-scale genome assembly of A. indica. The genome size of A. indica is 281 Mb anchored to 14 chromosomes (contig N50=6 Mb and scaffold N50=19 Mb). The genome assembly contained 115 Mb repetitive elements and 25,767 protein-coding genes. Evolutional analysis revealed that A. indica did not experience any whole-genome duplication (WGD) event after the core eudicot γ event, but some genes and genome segment might undergo recent duplications. The secondary metabolite clusters, TPS genes, and CYP genes were also identified. Comparative genomic analysis revealed that most of the A. indica-specific TPS genes and CYP genes were located on the terpene-related clusters on chromosome 13. It is suggested that chromosome 13 may play an important role in the specific terpene biosynthesis of A. indica. And the gene duplication events may be responsible for the terpene biosynthesis expansion in A. indica. This will shed light on terpene biosynthesis in A. indica and facilitate comparative genomic research of the family Meliaceae.

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