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Molecular Systems Biology 2006We have systematically made a set of precisely defined, single-gene deletions of all nonessential genes in Escherichia coli K-12. Open-reading frame coding regions were...
We have systematically made a set of precisely defined, single-gene deletions of all nonessential genes in Escherichia coli K-12. Open-reading frame coding regions were replaced with a kanamycin cassette flanked by FLP recognition target sites by using a one-step method for inactivation of chromosomal genes and primers designed to create in-frame deletions upon excision of the resistance cassette. Of 4288 genes targeted, mutants were obtained for 3985. To alleviate problems encountered in high-throughput studies, two independent mutants were saved for every deleted gene. These mutants-the 'Keio collection'-provide a new resource not only for systematic analyses of unknown gene functions and gene regulatory networks but also for genome-wide testing of mutational effects in a common strain background, E. coli K-12 BW25113. We were unable to disrupt 303 genes, including 37 of unknown function, which are candidates for essential genes. Distribution is being handled via GenoBase (http://ecoli.aist-nara.ac.jp/).
Topics: Escherichia coli; Gene Deletion; Internet; Mutation; Organisms, Genetically Modified
PubMed: 16738554
DOI: 10.1038/msb4100050 -
Methods in Molecular Biology (Clifton,... 2019Inactivation or deletion of genes allows for investigation and understanding of gene function. To facilitate markerless gene deletion in Listeria monocytogenes, we...
Inactivation or deletion of genes allows for investigation and understanding of gene function. To facilitate markerless gene deletion in Listeria monocytogenes, we developed a new suicide plasmid (pHoss1). pHoss1 contains the pMAD backbone, the secY antisense cassette from pIMAY driven by an inducible Pxyl/tetO promoter, a heat-sensitive origin of replication, four unique restriction sites (SalI, EcoRI, SmaI, and NcoI), and erythromycin resistance gene. We demonstrated that pHoss1 is very efficient for introducing mutations into different L. monocytogenes strains. In this chapter, we include a brief description of pHoss1 and the method used for gene deletion in L. monocytogenes using pHoss1.
Topics: Gene Deletion; Gene Targeting; Genes, Bacterial; Humans; Listeria monocytogenes; Listeriosis; Mutagenesis; Mutation; Plasmids
PubMed: 31197718
DOI: 10.1007/978-1-4939-9570-7_15 -
Redox Biology Oct 2023Long-chain acyl-CoA synthetase (ACSL) 4 converts polyunsaturated fatty acids (PUFAs) into their acyl-CoAs and plays an important role in maintaining PUFA-containing...
Long-chain acyl-CoA synthetase (ACSL) 4 converts polyunsaturated fatty acids (PUFAs) into their acyl-CoAs and plays an important role in maintaining PUFA-containing membrane phospholipids. Here we demonstrated decreases in various kinds of PUFA-containing phospholipid species in ACSL4-deficient murine lung. We then examined the effects of ACSL4 gene deletion on lung injury by treating mice with two pulmonary toxic chemicals: paraquat (PQ) and methotrexate (MTX). The results showed that ACSL4 deficiency attenuated PQ-induced acute lung lesion and decreased mortality. PQ-induced lung inflammation and neutrophil migration were also suppressed in ACSL4-deficient mice. PQ administration increased the levels of phospholipid hydroperoxides in the lung, but ACSL4 gene deletion suppressed their increment. We further found that ACSL4 deficiency attenuated MTX-induced pulmonary fibrosis. These results suggested that ACSL4 gene deletion might confer protection against pulmonary toxic chemical-induced lung injury by reducing PUFA-containing membrane phospholipids, leading to the suppression of lipid peroxidation. Inhibition of ACSL4 may be promising for the prevention and treatment of chemical-induced lung injury.
Topics: Mice; Animals; Lipid Peroxidation; Lung Injury; Xenobiotics; Gene Deletion; Phospholipids; Fatty Acids, Unsaturated; Lung; Ligases
PubMed: 37586249
DOI: 10.1016/j.redox.2023.102850 -
Methods in Molecular Biology (Clifton,... 2017Targeted gene deletion is a useful tool for understanding the function of a gene and its protein product. We have developed an efficient and robust gene deletion...
Targeted gene deletion is a useful tool for understanding the function of a gene and its protein product. We have developed an efficient and robust gene deletion approach in yeast that employs oligonucleotide-based gene synthesis. This approach requires a deletion cassette composed of three modules: a central 1397-bp KanMX4 selection marker module and two 366-bp gene-specific flanking modules. The invariable KanMX4 module can be used in combination with different pairs of flanking modules targeting different genes. The two flanking modules consist of both sequences unique to each cassette (chromosomal homologous regions and barcodes) and those common to all deletion constructs (artificial linkers and restriction enzyme sites). Oligonucleotides for each module and junction regions are designed using the BatchBlock2Oligo program and are synthesized on a 96-well basis. The oligonucleotides are ligated into a single deletion cassette by ligase chain reaction, which is then amplified through two rounds of nested PCR to obtain sufficient quantities for yeast transformation. After removal of the artificial linkers, the deletion cassettes are transformed into wild-type diploid fission yeast SP286 cells. Verification of correct clone and gene deletion is achieved by performing check PCR and tetrad analysis. This method with proven effectiveness, as evidenced by a high success rate of gene deletion, can be potentially applicable to create systematic gene deletion libraries in a variety of yeast species.
Topics: Base Sequence; Gene Deletion; Gene Targeting; Genes, Fungal; Mutagenesis, Insertional; Oligonucleotides; Schizosaccharomyces; Transformation, Genetic
PubMed: 27671940
DOI: 10.1007/978-1-4939-6343-0_13 -
Current Microbiology Jan 2023Sinorhizobium meliloti Rm1021 (S. meliloti Rm1021) is a Gram-negative, soil-dwelling α-proteobacterium which serves as a model microorganism for the studies of...
Sinorhizobium meliloti Rm1021 (S. meliloti Rm1021) is a Gram-negative, soil-dwelling α-proteobacterium which serves as a model microorganism for the studies of symbiotic nitrogen fixation. The S. meliloti Rm1021 genome consists of one chromosome and two megaplasmids, pSymA and pSymB. Gene deletion is an essential tool for the elucidation of gene function and generation of mutants with improved properties. However, only two gene deletion methods, counterselectable marker sacB-based and FLP/FRT, Cre/loxP site-specific recombination, have been reported for S. meliloti Rm1021 gene deletion. Both methods require time-consuming and tedious gene cloning and conjugation steps. Herein, a λ Red recombineering-mediated gene deletion strategy is reported. The mutant was obtained via electroporating overlap-extension PCR-generated linear targeting DNA into Red-proficient cells. One gene each from the S. meliloti Rm1021 chromosome, megaplasmid SymA and pSymB was deleted, with deletion efficiency up to 100%. The straightforward and highly efficient recombineering procedure holds the promise to be a general gene manipulation method for S. meliloti Rm1021.
Topics: Sinorhizobium meliloti; Gene Deletion; Bacterial Proteins
PubMed: 36650293
DOI: 10.1007/s00284-023-03188-1 -
Applied Microbiology and Biotechnology Aug 2020The ability to genetically manipulate microorganisms has been essential for understanding their biology and metabolism. Targeted genome editing relies on highly...
The ability to genetically manipulate microorganisms has been essential for understanding their biology and metabolism. Targeted genome editing relies on highly efficient homologous recombination, and while this is readily observed in the yeast Saccharomyces cerevisiae, most non-conventional yeast species do not display this trait and remain recalcitrant to targeted editing methods. CRISPR-based editing can bypass the requirement for high levels of native homologous recombination, enabling targeted modification to be more broadly implemented. While genetic transformation has been reported previously in Brettanomyces bruxellensis, a yeast with broad biotechnological potential and responsible for significant economic losses during the production of fermented beverages, targeted editing approaches have not been reported. Here, we describe the use of an expression-free CRISPR-Cas9 system, in combination with gene transformation cassettes tailored for B. bruxellensis, to provide the means for targeted gene deletion in this species. Deletion efficiency was shown to be dependent on homologous flanking DNA length, with higher targeting efficiencies observed with cassettes containing longer flanking regions. In a diploid strain, it was not possible to delete multiple alleles in one step, with heterozygous deletants only obtained when using DNA cassettes with long flanking regions. However, stepwise transformations (using two different marker genes) were successfully used to delete both wild-type alleles. Thus, the approach reported here will be crucial to understand the complex physiology of B. bruxellensis. Key points • The use of CRISPR-Cas9 enables targeted gene deletion in Brettanomyces bruxellensis. • Homozygous diploid deletions are possible with step-wise transformations. • Deletion of SSU1 confirmed the role of this gene in sulphite tolerance.
Topics: Alleles; Biotechnology; Brettanomyces; CRISPR-Cas Systems; Gene Deletion; Genome, Fungal; Sulfites; Transformation, Genetic
PubMed: 32592028
DOI: 10.1007/s00253-020-10750-5 -
International Journal of Laboratory... Aug 2021At present, a variety of molecular detection methods are obtained to diagnose thalassemia accurately. Although exome sequencing or specific panels have been widely used...
INTRODUCTION
At present, a variety of molecular detection methods are obtained to diagnose thalassemia accurately. Although exome sequencing or specific panels have been widely used in clinical diagnosis of genetic diseases, the positive rate is about 25%-30%. Because the detection range is limited to exons and splice sites, and the read length is usually 100-150 bp, there are limitations in the detection of globin gene clusters with pseudogenes.
METHODS
In this study, seven thalassemia patients were selected to perform whole-genome sequencing (WGS) with long read at 400 bp to make accurate detection for thalassemia deletions. And we used PCR and Sanger sequencing to confirm the gene deletions in the patients.
RESULTS
WGS analysis detected a rare 172 kb deletion on the α-globin gene cluster at chr16: 57 009-330 001, 19 kb deletion at chr16: 215 396-234 699, 11 kb deletion at chr16:220 861-231 981; and 27 kb deletion on the β-globin gene deletion at chr11: 5 222 878-5 250 288, 21.4 kb deletion at chr11: 5 236 361-5 257 771, 78.9 kb deletion at chr11: 5 191 121-5 270 050. All the seven patients carried heterozygous deletions, including three in α-gene cluster, three in β-gene cluster, and one in both globin clusters.
CONCLUSION
Our results indicate that long-read WGS will be beneficial to the diagnosis of genetic diseases with pseudogenes or highly duplicated sequences and will enable clinical geneticists to inform high-risk couples and provide prenatal diagnosis.
Topics: Adult; Female; Gene Deletion; High-Throughput Nucleotide Sequencing; Humans; Male; Multigene Family; Thalassemia; Whole Genome Sequencing
PubMed: 33400392
DOI: 10.1111/ijlh.13452 -
Journal of Clinical Oncology : Official... Mar 1998Since its discovery as a CDKI (cyclin-dependent kinase inhibitor) in 1993, the tumor suppressor p16 (INK4A/MTS-1/CDKN2A) has gained widespread importance in cancer. The... (Review)
Review
Since its discovery as a CDKI (cyclin-dependent kinase inhibitor) in 1993, the tumor suppressor p16 (INK4A/MTS-1/CDKN2A) has gained widespread importance in cancer. The frequent mutations and deletions of p16 in human cancer cell lines first suggested an important role for p16 in carcinogenesis. This genetic evidence for a causal role was significantly strengthened by the observation that p16 was frequently inactivated in familial melanoma kindreds. Since then, a high frequency of p16 gene alterations were observed in many primary tumors. In human neoplasms, p16 is silenced in at least three ways: homozygous deletion, methylation of the promoter, and point mutation. The first two mechanisms comprise the majority of inactivation events in most primary tumors. Additionally, the loss of p16 may be an early event in cancer progression, because deletion of at least one copy is quite high in some premalignant lesions. p16 is a major target in carcinogenesis, rivaled in frequency only by the p53 tumor-suppressor gene. Its mechanism of action as a CDKI has been elegantly elucidated and involves binding to and inactivating the cyclin D-cyclin-dependent kinase 4 (or 6) complex, and thus renders the retinoblastoma protein inactive. This effect blocks the transcription of important cell-cycle regulatory proteins and results in cell-cycle arrest. Although p16 may be involved in cell senescence, the physiologic role of p16 is still unclear. Future work will focus on studies of the upstream events that lead to p16 expression and its mechanism of regulation, and perhaps lead to better therapeutic strategies that can improve the clinical course of many lethal cancers.
Topics: Animals; Cell Cycle; Gene Deletion; Gene Expression Regulation, Neoplastic; Genes, p16; Humans; Mutation; Tumor Cells, Cultured
PubMed: 9508208
DOI: 10.1200/JCO.1998.16.3.1197 -
Virus Research Jan 2022Synthetic biology has been applied countless times for the modification and improvement of bacterial strains and for the synthesis of products that do not exist in...
Synthetic biology has been applied countless times for the modification and improvement of bacterial strains and for the synthesis of products that do not exist in nature. Phages are natural predators of bacteria controlling their population levels; however, their genomes carry several genes with unknown functions. In this work, Bacteriophage Recombineering of Electroporated DNA was used to assess the influence of deletion of a single gene with unknown function in the overall replication parameters of Salmonella phage PVP-SE2. Deletion of ORF_01, transcribed immediately after infection, reduced both the latent and rise periods by 5 min in PVP-SE2ΔORF_01 compared to the wild-type phage. A direct consequence of the deletion led to a smaller progeny release per infected cell by the mutant compared to the wild-type phage. Despite the difference in growth characteristics, the mutant phage remained infective towards exponentially growing cells. The mutation engineered endured for at least ten passages, showing that there is no reversion back to the wild-type sequence. This study provides proof of concept that methodologies used for phage engineering should always be complemented by phage growth characterization to assess whether a mutation can trigger undesirable characteristics.
Topics: Bacteriophages; Gene Deletion; Salmonella Phages; Salmonella enteritidis; Sequence Analysis, DNA
PubMed: 34902446
DOI: 10.1016/j.virusres.2021.198654 -
IEEE/ACM Transactions on Computational... 2023When simulating genome-scale metabolite production using constraint-based metabolic networks, it is often necessary to find gene deletion strategies which lead to...
When simulating genome-scale metabolite production using constraint-based metabolic networks, it is often necessary to find gene deletion strategies which lead to growth-coupled production, which means that target metabolites are produced when cell growth is maximized. Existing methods are effective when the number of gene deletions is relatively small, but when the number of required gene deletions exceeds approximately 1% of whole genes, the time required for the calculation is often unfeasible. Therefore, a complementing algorithm that is effective even when the required number of gene deletions is approximately 1% to 5% of whole genes would be helpful because the number of deletable genes in a strain is increasing with advances in genetic engineering technology. In this study, the author developed an algorithm, TrimGdel, which first computes a strategy with many gene deletions that results in growth-coupled production and then gradually reduces the number of gene deletions while ensuring the original production rate and growth rate. The results of the computer experiments showed that TrimGdel can calculate stoichiometrically feasible gene deletion strategies, especially those whose sizes are 1 to 5% of whole genes, which lead to growth-coupled production of many target metabolites, which include useful vitamins such as biotin and pantothenate, for which existing methods could not.
Topics: Gene Deletion; Metabolic Networks and Pathways; Algorithms; Genetic Engineering; Genome
PubMed: 35731759
DOI: 10.1109/TCBB.2022.3185221