Erratum: BRaf signaling principles unveiled by large-scale human mutation analysis with a rapid lentivirus-based gene replacement method

ABSTRACTWith voriconazole (VRC) being approved as the first choice in treating invasive aspergillosis (IA) and its increasing use in treatment, a VRC-resistant strain ofAspergillus flavus, the second leading cause of IA afterAspergillus fumigatus, has emerged. The VRC-resistant strain ofA. flavuswas isolated for the first time from the surgical lung specimen of an IA patient with no response to VRC therapy. In order to ascertain the mechanism of VRC resistance, the azole target enzyme genes in this strain ofA. flavuswere cloned and sequenced, and 4 mutations generating amino acid residue substitutions were found in thecyp51Cgene. To further determine the role of this mutated gene for VRC resistance inA. flavus, anAgrobacterium tumefaciens-mediated gene replacement approach was applied. Consequently, the mutatedcyp51Cgene from thisA. flavusstrain was proven to confer the VRC resistance. Finally, to discern the one out of the four mutations in thecyp51Cgene that is responsible for contributing to VRC resistance, a site-directed gene mutagenesis procedure combined with a gene replacement method was performed. As a result, the T788G missense mutation in thecyp51Cgene was identified as responsible for VRC resistance inA. flavus. These findings indicated that the detection of this mutation inA. flavuscould serve as an indicator for physicians to avoid the use of VRC during IA treatment. Further comprehensive surveillance for antifungal susceptibility, as well as intensive study on the mechanism of azole resistance inA. flavuscausing IA, would be required to fully understand this mechanism.

Download Full-text

Mass spider silk production through targeted gene replacement in Bombyx mori

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1806805115 ◽

2018 ◽

Vol 115 (35) ◽

pp. 8757-8762 ◽

Cited By ~ 31

Author(s):

Jun Xu ◽

Qinglin Dong ◽

Ye Yu ◽

Baolong Niu ◽

Dongfeng Ji ◽

...

Keyword(s):

Bombyx Mori ◽

Large Scale ◽

Spider Silk ◽

Natural Fibers ◽

Gene Replacement ◽

Chain Gene ◽

Scale Production ◽

Transcription Activator ◽

Efficient System ◽

Silk Production

Spider silk is one of the best natural fibers and has superior mechanical properties. However, the large-scale harvesting of spider silk by rearing spiders is not feasible, due to their territorial and cannibalistic behaviors. The silkworm, Bombyx mori, has been the most well known silk producer for thousands of years and has been considered an ideal bioreactor for producing exogenous proteins, including spider silk. Previous attempts using transposon-mediated transgenic silkworms to produce spider silk could not achieve efficient yields, due to variable promoter activities and endogenous silk fibroin protein expression. Here, we report a massive spider silk production system in B. mori by using transcription activator-like effector nuclease-mediated homology-directed repair to replace the silkworm fibroin heavy chain gene (FibH) with the major ampullate spidroin-1 gene (MaSp1) in the spider Nephila clavipes. We successfully replaced the ∼16-kb endogenous FibH gene with a 1.6-kb MaSp1 gene fused with a 1.1-kb partial FibH sequence and achieved up to 35.2% chimeric MaSp1 protein amounts in transformed cocoon shells. The presence of the MaSp1 peptide significantly changed the mechanical characteristics of the silk fiber, especially the extensibility. Our study provides a native promoter-driven, highly efficient system for expressing the heterologous spider silk gene instead of the transposon-based, random insertion of the spider gene into the silkworm genome. Targeted MaSp1 integration into silkworm silk glands provides a paradigm for the large-scale production of spider silk protein with genetically modified silkworms, and this approach will shed light on developing new biomaterials.

Download Full-text

Ham-Wasserman Lecture

Hematology ◽

10.1182/asheducation-2002.1.1 ◽

2002 ◽

Vol 2002 (1) ◽

pp. 1-9 ◽

Cited By ~ 40

Author(s):

Pier M. Mannucci

Keyword(s):

Large Scale ◽

Coagulation Factors ◽

Gene Replacement ◽

Infectious Complications ◽

Inherited Disorders ◽

Secondary Tumors ◽

Human Immunodeficiency Viruses ◽

First Millennium ◽

Viii And Ix ◽

Bloodborne Infections

Abstract Known since the beginning of the first millennium, the hemophilias are among the most frequent inherited disorders of blood coagulation and definitely the most severe. In the 1970s, with the availability of concentrated preparations of the deficient coagulation factors VIII and IX and with the large-scale adoption of home treatment, hemophilia care became one of the most gratifying examples of successful secondary prevention of a chronic disease. Unfortunately, in the early 1980s it was recognized that factor concentrates prepared from plasma pooled from thousands of donors transmitted the hepatitis and the human immunodeficiency viruses. The scientific community reacted promptly to the devastation brought about by hepatitis and AIDS. The last 15 years of the second millennium have witnessed the development of methods that, when applied during concentrate manufacturing, inactivate viruses escaping the screening procedures. The adoption of these measures has reduced dramatically the risk of transmission of bloodborne infections. The production of recombinant factors and their availability for patients’ treatment epitomize progress in hemophilia care through DNA technology. Methods based on the polymerase chain reaction (PCR) have unraveled an array of gene lesions associated with hemophilia, permitting improved secondary control of the disease through carrier detection in women from affected families and prenatal termination of their affected male infants. This article will review the aforementioned areas of progress and discuss unresolved problems (such as treatment of patients with antibodies, the risk of new infectious complications, and the issue of secondary tumors). Hopes and expectations for further improvement in the third millennium and particularly the prospects of hemophilia cure though gene replacement therapy will also be mentioned.

Download Full-text

A markerless gene replacement method for B. amyloliquefaciens LL3 and its use in genome reduction and improvement of poly-γ-glutamic acid production

Applied Microbiology and Biotechnology ◽

10.1007/s00253-014-5824-2 ◽

2014 ◽

Vol 98 (21) ◽

pp. 8963-8973 ◽

Cited By ~ 25

Author(s):

Wei Zhang ◽

Weixia Gao ◽

Jun Feng ◽

Chi Zhang ◽

Yulian He ◽

...

Keyword(s):

Glutamic Acid ◽

Acid Production ◽

Gene Replacement ◽

Genome Reduction ◽

Replacement Method ◽

Glutamic Acid Production

Download Full-text

Gene replacement method for determining conditions in which Bacillus subtilis genes are essential or dispensable for cell viability

Applied Microbiology and Biotechnology ◽

10.1007/s00253-003-1502-5 ◽

2004 ◽

Vol 64 (3) ◽

pp. 382-386 ◽

Cited By ~ 8

Author(s):

H. Yakhnin ◽

P. Babitzke

Keyword(s):

Bacillus Subtilis ◽

Cell Viability ◽

Gene Replacement ◽

Replacement Method

Download Full-text

An Improved Single-Step Cloning Strategy Simplifies the Agrobacterium tumefaciens-Mediated Transformation (ATMT)-Based Gene-Disruption Method for Verticillium dahliae

Phytopathology ◽

10.1094/phyto-10-15-0280-r ◽

2016 ◽

Vol 106 (6) ◽

pp. 645-652 ◽

Cited By ~ 12

Author(s):

Sheng Wang ◽

Haiying Xing ◽

Chenlei Hua ◽

Hui-Shan Guo ◽

Jie Zhang

Keyword(s):

Agrobacterium Tumefaciens ◽

Verticillium Dahliae ◽

Gene Disruption ◽

Large Scale ◽

Gene Deletion ◽

Binary Vector ◽

Gene Replacement ◽

Single Step ◽

Selection Marker ◽

Individual Gene

The soilborne fungal pathogen Verticillium dahliae infects a broad range of plant species to cause severe diseases. The availability of Verticillium genome sequences has provided opportunities for large-scale investigations of individual gene function in Verticillium strains using Agrobacterium tumefaciens-mediated transformation (ATMT)-based gene-disruption strategies. Traditional ATMT vectors require multiple cloning steps and elaborate characterization procedures to achieve successful gene replacement; thus, these vectors are not suitable for high-throughput ATMT-based gene deletion. Several advancements have been made that either involve simplification of the steps required for gene-deletion vector construction or increase the efficiency of the technique for rapid recombinant characterization. However, an ATMT binary vector that is both simple and efficient is still lacking. Here, we generated a USER-ATMT dual-selection (DS) binary vector, which combines both the advantages of the USER single-step cloning technique and the efficiency of the herpes simplex virus thymidine kinase negative-selection marker. Highly efficient deletion of three different genes in V. dahliae using the USER-ATMT-DS vector enabled verification that this newly-generated vector not only facilitates the cloning process but also simplifies the subsequent identification of fungal homologous recombinants. The results suggest that the USER-ATMT-DS vector is applicable for efficient gene deletion and suitable for large-scale gene deletion in V. dahliae.

Download Full-text

Targeted Disruption of the α-Amylase Gene in the Hyperthermophilic Archaeon Sulfolobus solfataricus

Journal of Bacteriology ◽

10.1128/jb.185.2.482-488.2003 ◽

2003 ◽

Vol 185 (2) ◽

pp. 482-488 ◽

Cited By ~ 102

Author(s):

Penny Worthington ◽

Viet Hoang ◽

Francisco Perez-Pomares ◽

Paul Blum

Keyword(s):

Genetic Manipulation ◽

Amylase Activity ◽

Sulfolobus Solfataricus ◽

Protein A ◽

Gene Replacement ◽

Coding Sequence ◽

Replacement Method ◽

Tandem Mass Spectroscopy ◽

New Gene

ABSTRACT Sulfolobus solfataricus secretes an acid-resistant α-amylase (amyA) during growth on starch as the sole carbon and energy source. Synthesis of this activity is subject to catabolite repression. To better understand α-amylase function and regulation, the structural gene was identified and disrupted and the resulting mutant was characterized. Internal α-amylase peptide sequences obtained by tandem mass spectroscopy were used to identify the amyA coding sequence. Anti-α-amylase antibodies raised against the purified protein immunoprecipitated secreted α-amylase activity and verified the enzymatic identity of the sequenced protein. A new gene replacement method was used to disrupt the amyA coding sequence by insertion of a modified allele of the S. solfataricus lacS gene. PCR and DNA sequence analysis were used to characterize the altered amyA locus in the recombinant strain. The amyA::lacS mutant lost the ability to grow on starch, glycogen, or pullulan as sole carbon and energy sources. During growth on a non-catabolite-repressing carbon source with added starch, the mutant produced no detectable secreted amylase activity as determined by enzyme assay, plate assay, or Western blot analysis. These results clarify the biological role of the α-amylase and provide additional methods for the directed genetic manipulation of the S. solfataricus genome.

Download Full-text

Construction of Nontoxigenic Mutants of Nonproteolytic Clostridium botulinum NCTC 11219 by Insertional Mutagenesis and Gene Replacement

Applied and Environmental Microbiology ◽

10.1128/aem.03703-15 ◽

2016 ◽

Vol 82 (10) ◽

pp. 3100-3108 ◽

Cited By ~ 3

Author(s):

Charlien Clauwers ◽

Kristof Vanoirbeek ◽

Laurence Delbrassinne ◽

Chris W. Michiels

Keyword(s):

Validation Studies ◽

Clostridium Botulinum ◽

Food Challenge ◽

Basic Research ◽

Gene Replacement ◽

Safe Alternative ◽

Process Validation ◽

Content Type ◽

Replacement Method ◽

New Gene

ABSTRACTGroup II nonproteolyticClostridium botulinum(gIICb) strains are an important concern for the safety of minimally processed ready-to-eat foods, because they can grow and produce botulinum neurotoxin during refrigerated storage. The principles of control of gIICb by conventional food processing and preservation methods have been well investigated and translated into guidelines for the food industry; in contrast, the effectiveness of emerging processing and preservation techniques has been poorly documented. The reason is that experimental studies withC. botulinumare cumbersome because of biosafety and biosecurity concerns. In the present work, we report the construction of two nontoxigenic derivatives of the type E gIICb strain NCTC 11219. In the first strain, the botulinum toxin gene (bont/E) was insertionally inactivated with a retargeted intron using the ClosTron system. In the second strain,bont/Ewas exchanged for an erythromycin resistance gene using a new gene replacement strategy that makes use ofpyrEas a bidirectional selection marker. Growth under optimal and stressed conditions, sporulation efficiency, and spore heat resistance of the mutants were unaltered, except for small differences in spore heat resistance at 70°C and in growth at 2.3% NaCl. The mutants described in this work provide a safe alternative for basic research as well as for food challenge and process validation studies with gIICb. In addition, this work expands the clostridial genetic toolbox with a new gene replacement method that can be applied to replace any gene in gIICb and other clostridia.IMPORTANCEThe nontoxigenic mutants described in this work provide a safe alternative for basic research as well as for food challenge and process validation studies with psychrotrophicClostridium botulinum. In addition, this work expands the clostridial genetic toolbox with a new gene replacement method that can be applied to replace any gene in clostridia.

Download Full-text