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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.

ABSTRACT: Background:Translocated 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. Results:Double-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. Conclusion:In 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.

SUBMITTER: Takahashi T

PROVIDER: S-EPMC6171174 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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

Takahashi Tadashi T Ogawa Masahiro M Sato Atsushi A Koyama Yasuji Y

Fungal biology and biotechnology 20181003

<h4>Background</h4>Translocated 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 ...[more]

PMID: 30305913

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Project description:Copper ligands of the recombinant tyrosinase from the fungus Aspergillus oryzae expressed in Saccharomyces cerevisiae or Escherichia coli were identified by site-directed mutagenesis. The recombinant protyrosinases expressed in S. cerevisiae were assayed for catalytic activities of mono-oxygenase and L-dopa oxidase at pH 5.5 after acid shock at pH 3.0. Replacements of His-63, His-84, His-93, His-290, His-294, His-332 or His-333 with asparagine resulted in mutant enzymes exhibiting no activities. The site-directed mutant Cys82Ala showed that Cys-82 was also an essential residue for the activity. We obtained homogeneous preparations of activated tyrosinases from mutated thioredoxin fusion gene products expressed in E. coli by acid shock. The copper contents of engineered mutants and wild-type enzyme expressed in E. coli were determined by atomic absorption spectrophotometry. The wild-type enzyme contained 2 g-atoms of copper/mol of the subunit. The His63Asn, His84Asn, His93Asn, His290Asn, His294Asn, His332Asn, His333Asn or Cys82Ala substitution decreased copper binding by approx. 50%, indicating that the mutants contain only approx. 1 g-atom of copper/mol of the subunit. The five mutants His63Asn, His93Asn, His290Asn, His294Asn and Cys82Ala contain only one copper ion, which is fully detectable by EPR. From the correlation of g( parallel) and (Cu)A( parallel), we deduced that the nitrogen or sulphur donors in the copper ligands should be in a square or a distorted tetrahedral geometric environment. In further atomic absorption spectrophotometry experiments, no copper atom was observed in the seven double mutants His63Asn/His290Asn, His63Asn/His294Asn, His63Asn/His332Asn, His63Asn/His333Asn, Cys82Ala/His290Asn, His84Asn/His333Asn and His93Asn/His290Asn. We propose a new structure of active sites of tyrosinase from A. oryzae: the most likely binding sites of tyrosinase for Cu(A) are His-63, His-84 and His-93, with the remaining conserved Cys-82 providing the fourth ligand. Cu(B) liganded by four histidine residues, His-290, His-294, His-332 and His-333, is identified as new binding motif of Cu(B).

Dataset Information

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

Publications

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

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