Targeted Tandem Duplication of a Large Chromosomal Segment in Aspergillus oryzae

Takahashi, Tadashi; Sato, Atsushi; Ogawa, Mitsuhiro; Hanya, Yoshiki; Oguma, Tetsuya

doi:10.1128/aem.00300-14

Cited by 4 publications

(18 citation statements)

References 26 publications

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“…We previously constructed strains containing targeted tandem chromosomal duplications [ 12 ]. Chromosome 2 of A. oryzae includes genes encoding alkaline protease and alpha-amylase, and their respective regulatory genes prtT and amyR , which are important for fermentation.…”

Section: Resultsmentioning

confidence: 99%

“…Overexpression of individual genes via a strong promoter can also cause reduced growth phenotypes [ 11 ]. However, strains with duplicated chromosomes can show phenotypic changes via increased dosage of unidentified genes [ 12 ]. By examining the genome of targeted chromosomal segments and phenotypic changes in the strains, we can determine the role of unknown genes corresponding to these phenotypic changes.…”

Section: Introductionmentioning

confidence: 99%

“…1 , bottom). We previously generated A. oryzae strains containing targeted tandem chromosomal duplications using protoplasts of the parental strain, in which artificially generated consensus sequences (partly deleted selection marker) were introduced into both ends of the duplication target region [ 12 ]. If the copy number of the duplication target region is increased, such as in triplication, the phenotypic change in the strain may be further enhanced.…”

Section: Introductionmentioning

confidence: 99%

“…However, to maintain stability of a tandem chromosomal duplication, a selection marker must be located at the junction between the two duplicated segments. In A. oryzae , this prevents removal of the duplicated region by recombination events occurring between the duplicated homologous sequences [ 12 ]. Therefore, it is technically difficult to increase the copy numbers of genes located in duplicated regions in tandem chromosomal duplications.…”

Section: Introductionmentioning

confidence: 99%

“…In most studies on yeast, DSBs are introduced after the expression of genes encoding endogenous yeast homothallic switching endonuclease ( HO ) or I-SceI endonuclease ( SCEI ). In contrast, chromosome modifications have been performed in A. oryzae using polyethylene glycol (PEG)-mediated introduction of enzymes into protoplast cells [ 12 , 16 – 18 ]. Therefore, we generated chromosomal duplications by directly treating protoplast cells with I-SceI meganuclease and monitoring the resulting chromosomal DSBs.…”

Section: Introductionmentioning

confidence: 99%

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Translocated duplication of a targeted chromosomal segment enhances gene expression at the duplicated site and results in phenotypic changes in Aspergillus oryzae

Takahashi

Ogawa

Sato

et al. 2018

Fungal Biol Biotechnol

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BackgroundTranslocated chromosomal duplications occur spontaneously in many organisms; segmental duplications of large chromosomal regions are expected to result in phenotypic changes because of gene dosage effects. Therefore, experimentally generated segmental duplications in targeted chromosomal regions can be used to study phenotypic changes and determine the functions of unknown genes in these regions. Previously, we performed tandem duplication of a targeted chromosomal segment in Aspergillus oryzae. However, in tandem chromosomal duplication, duplication of chromosomal ends and multiple chromosomal duplication are difficult. In this study, we aimed to generate fungal strains with a translocated duplication or triplication of a targeted chromosomal region via break-induced replication.ResultsDouble-strand breaks were introduced into chromosomes of parental strains by treating protoplast cells with I-SceI meganuclease. Subsequently, strains were generated by nonreciprocal translocation of a 1.4-Mb duplicated region of chromosome 2 to the end of chromosome 4. Another strain, containing a triplicated region of chromosome 2, was generated by translocating a 1.4-Mb region of chromosome 2 onto the ends of chromosomes 4 and 7. Phenotypic analyses of the strains containing segmental duplication or triplication of chromosome 2 showed remarkable increases in protease and amylase activities in solid-state cultures. Protease activity was further increased in strains containing the duplication and triplication after overexpression of the transcriptional activator of proteases prtT. This indicates that the gene-dosage effect and resulting phenotypes of the duplicated chromosomal region were enhanced by multiple duplications, and by the combination of the structural gene and its regulatory genes. Gene expression analysis, conducted using oligonucleotide microarrays, showed increased transcription of a large population of genes located in duplicated or triplicated chromosomal regions.ConclusionIn this study, we performed translocated chromosomal duplications and triplications of a 1.4-Mb targeted region of chromosome 2. Strains containing a duplication of chromosome 2 showed significant increases in protease and amylase activities; these enzymatic activities were further increased in the strain containing a triplication of chromosome 2. This indicates that segmental duplications of chromosomes enhance gene-dosage effects, and that the resulting phenotypes play important phenotypic roles in A. oryzae.Electronic supplementary materialThe online version of this article (10.1186/s40694-018-0061-6) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%