High frequency one-step gene replacement in Trichoderma reesei. I. Endoglucanase I overproduction

1993 ◽  
Vol 241-241 (5-6) ◽  
pp. 515-522 ◽  
Author(s):  
Taina Karhunen ◽  
Arja Mäntylä ◽  
K. M. Helena Nevalainen ◽  
Pirkko L. Suominen
Keyword(s):  
Trichoderma Reesei ◽  
High Frequency ◽  
Gene Replacement ◽  
One Step ◽  
Endoglucanase I

High frequency one-step gene replacement in Trichoderma reesei. II. Effects of deletions of individual cellulase genes

1993 ◽  
Vol 241-241 (5-6) ◽  
pp. 523-530 ◽  
Author(s):  
Pirkko L. Suominen ◽  
Arja L. Mäntylä ◽  
Taina Karhunen ◽  
Satu Hakola ◽  
Helena Nevalainen
Keyword(s):  
Trichoderma Reesei ◽  
High Frequency ◽  
Gene Replacement ◽  
One Step

High-frequency homologous recombination between duplicate chromosomal immunoglobulin mu heavy-chain constant regions

1989 ◽  
Vol 9 (12) ◽  
pp. 5500-5507
Author(s):  
M D Baker
Keyword(s):  
Homologous Recombination ◽  
Heavy Chain ◽  
High Frequency ◽  
Gene Replacement ◽  
Immunoglobulin M ◽  
Hybridoma Cells ◽  
Immunoglobulin Gene ◽  
Reciprocal Recombination ◽  
Base Pair Deletion ◽  
Frequent Event

Homologous recombination was used in a previous study to correct a 2-base-pair deletion in the third constant domain (Cmu3) of the haploid chromosomal mu gene in a mutant hybridoma cell line by transfer of a pSV2neo vector bearing a subfragment of the normal Cmu region (M.D. Baker, N. Pennell, L. Bosnoyan, and M.J. Shulman, Proc. Natl. Acad. Sci. USA 85:6432-6436, 1988). In these experiments, both gene replacement and single reciprocal crossover events were found to restore normal, cytolytic 2,4,6-trinitrophenyl-specific immunoglobulin M production to the mutant cells. In the cases of single reciprocal recombination, the structure of the recombinant mu gene is such that the normal Cmu region, in its correct position 3' of the expressed 2,4,6-trinitrophenyl-specific heavy-chain variable region, is separated from the mutant Cmu region by the integrated vector sequences. I report here that homologous recombination occurs with high frequency between the duplicate Cmu regions in mitotically growing hybridoma cells. The homologous recombination events were easily detected since they generated hybridomas that were phenotypically different from the parental cells. Analysis of the recombinant cells suggests that gene conversion is the most frequent event, occurring between 60 and 73% of the time. The remaining events consisted of single reciprocal crossovers. Intrachromatid double reciprocal recombination was not detected. The high frequency of recombination, the ability to isolate and analyze the participants in the recombination reactions, and the capacity to generate specific modifications in the immunoglobulin Cmu regions by gene targeting suggest that this system will be useful for studying mammalian chromosomal homologous recombination. Moreover, the ability to specifically modify the chromosomal immunoglobulin genes by homologous recombination should facilitate studies of immunoglobulin gene regulation and expression and provide a more convenient of engineering specifically modified antibody.

scholarly journals Gene Replacement in Mycobacteria by Using Incompatible Plasmids

2003 ◽  
Vol 69 (1) ◽  
pp. 517-523 ◽  
Author(s):  
Carey A. Pashley ◽  
Tanya Parish ◽  
Ruth A. McAdam ◽  
Ken Duncan ◽  
Neil G. Stoker
Keyword(s):  
Delivery System ◽  
High Frequency ◽  
Mycobacterium Smegmatis ◽  
Plasmid Vector ◽  
Gene Replacement ◽  
Replication Machinery ◽  
Daughter Cells ◽  
Simple Addition ◽  
Efficient Delivery ◽  
Gene Knockouts

ABSTRACT A simple and efficient delivery system was developed for making targeted gene knockouts in Mycobacterium smegmatis. This delivery system relies on the use of a pair of replicating plasmids, which are incompatible. Incompatible plasmids share elements of the same replication machinery and so compete with each other during both replication and partitioning into daughter cells. Such plasmids can be maintained together in the presence of antibiotics; however, removal of selection leads to the loss of one or both plasmids. For mutagenesis, two replicating plasmids based on pAL5000 are introduced; one of these plasmids carries a mutated allele of the targeted gene. Homologous recombination is allowed to take place, and either one or both of the vectors are lost through the pressure of incompatibility, allowing the phenotypic effects of the mutant to be studied. Several different plasmid combinations were tested to optimize loss in the absence of antibiotic selection. pAL5000 carries two replication genes (repA and repB), which act in trans, and the use of vectors that each lack one rep gene and complement each other resulted in the loss of both plasmids in M. smegmatis and Mycobacterium bovis BCG. The rate of loss was increased by the incorporation of an additional incompatibility region in one of the plasmids. To facilitate cloning when the system was used, we constructed plasmid vector pairs that allow simple addition of selection and screening genes on flexible gene cassettes. Using this system, we demonstrated that M. smegmatis pyrF mutants could be isolated at high frequency. This method should also be useful in other species in which pAL5000 replicates, including Mycobacterium tuberculosis.

The crystal structure of the catalytic core domain of endoglucanase I from Trichoderma reesei at 3.6 Å resolution, and a comparison with related enzymes 1 1Edited by K.Nagai

1997 ◽  
Vol 272 (3) ◽  
pp. 383-397 ◽  
Author(s):  
Gerard J Kleywegt ◽  
Jin-Yu Zou ◽  
Christina Divne ◽  
Gideon J Davies ◽  
Irmgard Sinning ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Trichoderma Reesei ◽  
Catalytic Core Domain ◽  
Catalytic Core ◽  
Core Domain ◽  
Endoglucanase I

Simultaneous Synthesis and Consolidation of Nanostructured ZrB2–ZrO2 Composite

2020 ◽  
Vol 20 (7) ◽  
pp. 4349-4352
Author(s):  
Seong-Eun Kim ◽  
Jin-Kook Yoon ◽  
In-Jin Shon
Keyword(s):  
Mechanical Properties ◽  
Combustion Synthesis ◽  
High Frequency ◽  
Combined Effects ◽  
Induced Current ◽  
Mechanical Pressure ◽  
Simultaneous Application ◽  
Average Grain Size ◽  
One Step ◽  
Simultaneous Synthesis

A dense nanostructured 2ZrB2–ZrO2 composite was synthesized by the high-frequency inductionheated combustion synthesis (HFIHCS) method within 2 min in one step from mechanically activated powders of 2B2O3 and 3Zr. Simultaneous combustion synthesis and densification were accomplished under the combined effects of the induced current and mechanical pressure. A highly dense 2ZrB2–ZrO2 composite with relative density of up to 95.5% was produced under the simultaneous application of a pressure of 80 MPa and the induced current. The average grain size and mechanical properties (hardness and fracture toughness) of the composite were investigated.

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