scholarly journals Isolation and Molecular Identification of Auxotrophic Mutants to Develop a Genetic Manipulation System for the HaloarchaeonNatrinemasp. J7-2

Archaea ◽  
2015 ◽  
Vol 2015 ◽  
pp. 1-16 ◽  
Author(s):  
Jie Lv ◽  
Shuai Wang ◽  
Yuchen Wang ◽  
Yuping Huang ◽  
Xiangdong Chen
Keyword(s):  
Genomic Dna ◽  
Genetic Manipulation ◽  
Gene Mutations ◽  
Transformation Method ◽  
Wild Type ◽  
Auxotrophic Mutants ◽  
Archaeal Species ◽  
Manipulation System ◽  
Exogenous Genes ◽  
Mutant Phenotypes

Our understanding of the genusNatrinemais presently limited due to the lack of available genetic tools. Auxotrophic markers have been widely used to construct genetic systems in bacteria and eukaryotes and in some archaeal species. Here, we isolated four auxotrophic mutants ofNatrinemasp. J7-2, via 1-methyl-3-nitro-1-nitroso-guanidin mutagenesis, and designated them as J7-2-1, J7-2-22, J7-2-26, and J7-2-52, respectively. The mutant phenotypes were determined to be auxotrophic for leucine (J7-2-1), arginine (J7-2-22 and J7-2-52), and lysine (J7-2-26). The complete genome and the biosynthetic pathways of amino acids in J7-2 identified that the auxotrophic phenotype of three mutants was due to gene mutations inleuB(J7-2-1),dapD(J7-2-26), andargC(J7-2-52). These auxotrophic phenotypes were employed as selectable makers to establish a transformation method. The transformation efficiencies were determined to be approximately 103transformants perµg DNA. And strains J7-2-1 and J7-2-26 were transformed into prototrophic strains with the wild type genomic DNA, amplified fragments of the corresponding genes, or the integrative plasmids carrying the corresponding genes. Additionally, exogenous genes,bgaHoramyHgene, were expressed successfully in J7-2-1. Thus, we have developed a genetic manipulation system for theNatrinemagenus based on the isolated auxotrophic mutants ofNatrinemasp. J7-2.

Using 16S rDNA as Target Site for Homologous Recombination to Improve the Alkaline Protease Production of Bacillus alcalophilus

2014 ◽  
Vol 886 ◽  
pp. 349-354
Author(s):  
Qing Shan Mo ◽  
Yao Tian ◽  
Hui Tu Zhang ◽  
Ling Jun Bu ◽  
Fu Ping Lu
Keyword(s):  
16S Rdna ◽  
Alkaline Protease ◽  
Genetic Manipulation ◽  
Protease Production ◽  
Protease Gene ◽  
Wild Type ◽  
Recombinant Strains ◽  
Manipulation System ◽  
Rdna Sites ◽  
Alkaline Protease Gene

Bacillus alcalophilusisolated was used for the production of alkaline protease. The enzyme encoded by alkaline protease gene (apr4) gene. To further improve the production of the strain for industrial requirement, a genetic manipulation system forBacillus alcalophiluswas developed. Additional copies of theapr4 gene were transferred into the strainBacillus alcalophilusand integrated into the 16S rDNA sites, yielding a series of recombinant strains. One of these recombinant strains, designated K23, exhibited superior properties for production of alkaline protease. the protease activity of K23 achieved by (6.19 ± 0.34) × 104U/ml, which is approximately 111.3% higher than that of the wild-type ones for 50-h fermentation. In addition, the new strain was genetically stable for more than 100 generations. These superior characteristics make it to be more suitable than the wild-type strain for alkaline protease production.

scholarly journals Establishing an Efficient Genetic Manipulation System for Sulfated Echinocandin Producing Fungus Coleophoma empetri

2021 ◽  
Vol 12 ◽  
Author(s):  
Ping Men ◽  
Min Wang ◽  
Jinda Li ◽  
Ce Geng ◽  
Xuenian Huang ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Secondary Metabolites ◽  
Polyketide Synthase ◽  
Genetic Manipulation ◽  
Fungal Infections ◽  
Genome Mining ◽  
Transformation Method ◽  
Selectable Markers ◽  
Cyclic Hexapeptide ◽  
Manipulation System

Micafungin is an important echinocandin antifungal agent for the treatment of invasive fungal infections. In industry, micafungin is derived from the natural product FR901379, which is a non-ribosomal cyclic hexapeptide produced by the filamentous fungus Coleophoma empetri. The difficulty of genetic manipulation in C. empetri restricts the clarification of FR901379 biosynthetic mechanism. In this work, we developed an efficient genetic manipulation system in the industrial FR901379-producing strain C. empetri MEFC009. Firstly, a convenient protoplast-mediated transformation (PMT) method was developed. Secondly, with this transformation method, the essential genetic elements were verified. Selectable markers hph, neo, and nat can be used for the transformation, and promotors Ppgk, PgpdA, and PgpdAt are functional in C. empetri MEFC009. Thirdly, the frequency of homologous recombination was improved from 4 to 100% by deleting the ku80 gene, resulting in an excellent chassis cell for gene-targeting. Additionally, the advantage of this genetic manipulation system was demonstrated in the identification of the polyketide synthase (PKS) responsible for the biosynthesis of dihydroxynapthalene (DHN)-melanin. This genetic manipulation system will be a useful platform for the research of FR901379 and further genome mining of secondary metabolites in C. empetri.

scholarly journals Allelic Exchange and Mutant Selection Demonstrate that Common Clinical embCAB Gene Mutations Only Modestly Increase Resistance to Ethambutol in Mycobacterium tuberculosis

2009 ◽  
Vol 54 (1) ◽  
pp. 103-108 ◽  
Author(s):  
Hassan Safi ◽  
Robert D. Fleischmann ◽  
Scott N. Peterson ◽  
Marcus B. Jones ◽  
Behnam Jarrahi ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
In Vitro Selection ◽  
Gene Mutations ◽  
Wild Type ◽  
Mutant Selection ◽  
Preferential Growth ◽  
Allelic Exchange ◽  
High Level ◽  
Isogenic Mutants

ABSTRACT Mutations within codon 306 of the Mycobacterium tuberculosis embB gene modestly increase ethambutol (EMB) MICs. To identify other causes of EMB resistance and to identify causes of high-level resistance, we generated EMB-resistant M. tuberculosis isolates in vitro and performed allelic exchange studies of embB codon 406 (embB406) and embB497 mutations. In vitro selection produced mutations already identified clinically in embB306, embB397, embB497, embB1024, and embC13, which result in EMB MICs of 8 or 14 μg/ml, 5 μg/ml, 12 μg/ml, 3 μg/ml, and 4 μg/ml, respectively, and mutations at embB320, embB324, and embB445, which have not been identified in clinical M. tuberculosis isolates and which result in EMB MICs of 8 μg/ml, 8 μg/ml, and 2 to 8 μg/ml, respectively. To definitively identify the effect of the common clinical embB497 and embB406 mutations on EMB susceptibility, we created a series of isogenic mutants, exchanging the wild-type embB497 CAG codon in EMB-susceptible M. tuberculosis strain 210 for the embB497 CGG codon and the wild-type embB406 GGC codon for either the embB406 GCC, embB406 TGC, embB406 TCC, or embB406 GAC codon. These new mutants showed 6-fold and 3- to 3.5-fold increases in the EMB MICs, respectively. In contrast to the embB306 mutants, the isogenic embB497 and embB406 mutants did not have preferential growth in the presence of isoniazid or rifampin (rifampicin) at their MICs. These results demonstrate that individual embCAB mutations confer low to moderate increases in EMB MICs. Discrepancies between the EMB MICs of laboratory mutants and clinical M. tuberculosis strains with identical mutations suggest that clinical EMB resistance is multigenic and that high-level EMB resistance requires mutations in currently unknown loci.

scholarly journals The Mutant Phenotype Associated With P-Element Alleles of the vestigial Locus in Drosophila melanogaster May Be Caused by a Readthrough Transcript Initiated at the P-Element Promoter

Genetics ◽  
2001 ◽  
Vol 157 (4) ◽  
pp. 1665-1672 ◽  
Author(s):  
Ross B Hodgetts ◽  
Sandra L O'Keefe
Keyword(s):  
Rna Secondary Structure ◽  
Insertion Site ◽  
Large Body ◽  
P Element ◽  
Wild Type ◽  
Subtle Difference ◽  
Pronounced Difference ◽  
Internal Repeat ◽  
Mutant Phenotypes

Abstract We report here the isolation of a new P-element-induced allele of the vestigial locus vg2a33, the molecular characterization of which allows us to propose a unifying explanation of the phenotypes of the large number of vestigial P-element alleles that now exists. The first P-element allele of vestigial to be isolated was vg21, which results in a very weak mutant wing phenotype that is suppressed in the P cytotype. By destabilizing vg2a33 in a dysgenic cross, we isolated the vg2a33 allele, which exhibits a moderate mutant wing phenotype and is not suppressed by the P cytotype. The new allele is characterized by a 46-bp deletion that removes the 3′-proximal copy of the 11-bp internal repeat from the P element of vg21. To understand how this subtle difference between the two alleles leads to a rather pronounced difference in their phenotypes, we mapped both the vg and P-element transcription units present in wild type and mutants. Using both 5′-RACE and S1 protection, we found that P-element transcription is initiated 19 bp farther upstream than previously thought. Using primer extension, the start of vg transcription was determined to lie 435 bp upstream of the longest cDNA recovered to date and upstream of the P-element insertion site. Our discovery that the P element is situated within the first vg exon has prompted a reassessment of the large body of genetic data on a series of alleles derived from vg21. Our current hypothesis to explain the degree of variation in the mutant phenotypes and their response to the P repressor invokes a critical RNA secondary structure in the vg transcript, the formation of which is hindered by a readthrough transcript initiated at the P-element promoter.

scholarly journals FGFR3 overexpression is a useful detection tool for FGFR3 fusions and sequence variations in glioma

2020 ◽  
Author(s):  
Jens Schittenhelm ◽  
Lukas Ziegler ◽  
Jan Sperveslage ◽  
Michel Mittelbronn ◽  
David Capper ◽  
...  
Keyword(s):  
Coiled Coil ◽  
Growth Factor Receptor ◽  
Gene Mutations ◽  
Diagnostic Tools ◽  
Wild Type ◽  
Rt Pcr ◽  
Fgfr3 Gene ◽  
Detection Tool ◽  
Sequence Variations ◽  
Gene Panel Sequencing

Abstract Background Fibroblast growth factor receptor (FGFR) inhibitors are currently used in clinical development. A subset of glioblastomas carries gene fusion of FGFR3 and transforming acidic coiled-coil protein 3. The prevalence of other FGFR3 alterations in glioma is currently unclear. Methods We performed RT-PCR in 101 glioblastoma samples to detect FGFR3-TACC3 fusions (“RT-PCR cohort”) and correlated results with FGFR3 immunohistochemistry (IHC). Further, we applied FGFR3 IHC in 552 tissue microarray glioma samples (“TMA cohort”) and validated these results in two external cohorts with 319 patients. Gene panel sequencing was carried out in 88 samples (“NGS cohort”) to identify other possible FGFR3 alterations. Molecular modeling was performed on newly detected mutations. Results In the “RT-PCR cohort,” we identified FGFR3-TACC3 fusions in 2/101 glioblastomas. Positive IHC staining was observed in 73/1024 tumor samples of which 10 were strongly positive. In the “NGS cohort,” we identified FGFR3 fusions in 9/88 cases, FGFR3 amplification in 2/88 cases, and FGFR3 gene mutations in 7/88 cases in targeted sequencing. All FGFR3 fusions and amplifications and a novel FGFR3 K649R missense mutation were associated with FGFR3 overexpression (sensitivity and specificity of 93% and 95%, respectively, at cutoff IHC score > 7). Modeling of these data indicated that Tyr647, a residue phosphorylated as a part of FGFR3 activation, is affected by the K649R mutation. Conclusions FGFR3 IHC is a useful screening tool for the detection of FGFR3 alterations and could be included in the workflow for isocitrate dehydrogenase (IDH) wild-type glioma diagnostics. Samples with positive FGFR3 staining could then be selected for NGS-based diagnostic tools.

scholarly journals Mutants of downy mildew resistance in Lactuca sativa (lettuce).

Genetics ◽  
1994 ◽  
Vol 137 (3) ◽  
pp. 867-874
Author(s):  
P A Okubara ◽  
P A Anderson ◽  
O E Ochoa ◽  
R W Michelmore
Keyword(s):  
Molecular Marker ◽  
Downy Mildew ◽  
Genomic Dna ◽  
Spontaneous Mutation ◽  
Multigene Families ◽  
Downy Mildew Resistance ◽  
Bremia Lactucae ◽  
Wild Type ◽  
Mildew Resistance ◽  
Recessive Mutations

Abstract As part of our investigation of disease resistance in lettuce, we generated mutants that have lost resistance to Bremia lactucae, the casual fungus of downy mildew. Using a rapid and reliable screen, we identified 16 distinct mutants of Latuca sativa that have lost activity of one of four different downy mildew resistance genes (Dm). In all mutants, only a single Dm specificity was affected. Genetic analysis indicated that the lesions segregated as single, recessive mutations at the Dm loci. Dm3 was inactivated in nine of the mutants. One of five Dm 1 mutants was selected from a population of untreated seeds and therefore carried a spontaneous mutation. All other Dm1, Dm3, Dm5/8 and Dm7 mutants were derived from gamma- or fast neutron-irradiated seed. In two separate Dm 1 mutants and in each of the eight Dm3 mutants analyzed, at least one closely linked molecular marker was absent. Also, high molecular weight genomic DNA fragments that hybridized to a tightly linked molecular marker in wild type were either missing entirely or were truncated in two of the Dm3 mutants, providing additional evidence that deletions had occurred in these mutants. Absence of mutations at loci epistatic to the Dm genes suggested that such loci were either members of multigene families, were critical for plant survival, or encoded components of duplicated pathways for resistance; alternatively, the genes determining downy mildew resistance might be limited to the Dm loci.

scholarly journals Analysis of maxillopedia Expression Pattern and Larval Cuticular Phenotype in Wild-Type and Mutant Tribolium

Genetics ◽  
2000 ◽  
Vol 155 (2) ◽  
pp. 721-731 ◽  
Author(s):  
Teresa D Shippy ◽  
Jianhua Guo ◽  
Susan J Brown ◽  
Richard W Beeman ◽  
Robin E Denell
Keyword(s):  
Rna Interference ◽  
Expression Pattern ◽  
Tribolium Castaneum ◽  
Expression Patterns ◽  
Ectopic Expression ◽  
Homeotic Gene ◽  
Wild Type ◽  
Double Stranded Rna ◽  
Similar Expression ◽  
Mutant Phenotypes

Abstract The Tribolium castaneum homeotic gene maxillopedia (mxp) is the ortholog of Drosophila proboscipedia (pb). Here we describe and classify available mxp alleles. Larvae lacking all mxp function die soon after hatching, exhibiting strong transformations of maxillary and labial palps to legs. Hypomorphic mxp alleles produce less severe transformations to leg. RNA interference with maxillopedia double-stranded RNA results in phenocopies of mxp mutant phenotypes ranging from partial to complete transformations. A number of gain-of-function (GOF) mxp alleles have been isolated based on transformations of adult antennae and/or legs toward palps. Finally, we have characterized the mxp expression pattern in wild-type and mutant embryos. In normal embryos, mxp is expressed in the maxillary and labial segments, whereas ectopic expression is observed in some GOF variants. Although mxp and Pb display very similar expression patterns, pb null embryos develop normally. The mxp mutant larval phenotype in Tribolium is consistent with the hypothesis that an ancestral pb-like gene had an embryonic function that was lost in the lineage leading to Drosophila.

Molecular Markers for Rapidly Identifying Candidate Genes in Chlamydomonas reinhardtii: ERY1 and ERY2 Encode Chloroplast Ribosomal Proteins

Genetics ◽  
2003 ◽  
Vol 164 (4) ◽  
pp. 1345-1353
Author(s):  
Amber K Bowers ◽  
Jennifer A Keller ◽  
Susan K Dutcher
Keyword(s):  
Molecular Markers ◽  
Chlamydomonas Reinhardtii ◽  
Candidate Genes ◽  
Splice Site ◽  
Genomic Dna ◽  
Ribosomal Proteins ◽  
Genomic Sequence ◽  
Point Mutations ◽  
Acceptor Site ◽  
Wild Type

Abstract To take advantage of available expressed sequence tags and genomic sequence, we have developed 64 PCR-based molecular markers in Chlamydomonas reinhardtii that map to the 17 linkage groups. These markers will allow the rapid association of a candidate gene sequence with previously identified mutations. As proof of principle, we have identified the genes encoded by the ERY1 and ERY2 loci. Mendelian mutations that confer resistance to erythromycin define three unlinked nuclear loci in C. reinhardtii. Candidate genes ribosomal protein L4 (RPL4) and L22 (RPL22) are tightly linked to the ERY1 locus and ERY2 locus, respectively. Genomic DNA for RPL4 from wild type and five mutant ery1 alleles was amplified and sequenced and three different point mutations were found. Two different glycine residues (G102 and G112) are replaced by aspartic acid and both are in the unstructured region of RPL4 that lines the peptide exit tunnel of the chloroplast ribosome. The other two alleles change a splice site acceptor site. Genomic DNA for RPL22 from wild type and three mutant ery2 alleles was amplified and sequenced and revealed three different point mutations. Two alleles have premature stop codons and one allele changes a splice site acceptor site.

scholarly journals Homology-Dependent Silencing of the SC3 Gene in Schizophyllum commune

Genetics ◽  
1997 ◽  
Vol 147 (2) ◽  
pp. 589-596 ◽  
Author(s):  
Theo A Schuurs ◽  
Eveline A M Schaeffer ◽  
Joseph G H Wessels
Keyword(s):  
Gene Silencing ◽  
Genomic Dna ◽  
Type Strain ◽  
Cytosine Methylation ◽  
Wild Type Strain ◽  
Schizophyllum Commune ◽  
Southern Analysis ◽  
Transcriptional Level ◽  
Wild Type

After introduction of extra copies of the SC3 hydrophobin gene into a wild-type strain of Schizophyllum commune, gene silencing was observed acting on both endogenous and introduced SC3 genes in primary vegetative transformants. Nuclear run-on experiments indicated that silencing acted at the transcriptional level. Southern analysis revealed that cytosine methylation of genomic DNA occurred. Moreover, SC3 silencing was suppressed by exposure to 5-azacytidine during growth. After growth of SC3-suppressed colonies from homogenized mycelium or from colonies stored at 4°, SC3 transcription was restored. However, after prolonged growth SC3 silencing was again observed. Introduction of a promoterless SC3 fragment into wild type gave less SC3 silencing.

scholarly journals Molecular Basis of In Vivo Resistance to Sulfadoxine-Pyrimethamine in African Adult Patients Infected withPlasmodium falciparum Malaria Parasites

1998 ◽  
Vol 42 (7) ◽  
pp. 1811-1814 ◽  
Author(s):  
Leonardo K. Basco ◽  
Rachida Tahar ◽  
Pascal Ringwald
Keyword(s):  
Dihydrofolate Reductase ◽  
Point Mutations ◽  
Gene Mutations ◽  
Adult Patients ◽  
Wild Type ◽  
Dihydropteroate Synthase ◽  
Reductase Gene ◽  
Parasite Populations

ABSTRACT In vitro sulfadoxine and pyrimethamine resistance has been associated with point mutations in the dihydropteroate synthase and dihydrofolate reductase domains, respectively, but the in vivo relevance of these point mutations has not been well established. To analyze the correlation between genotype and phenotype, 10 Cameroonian adult patients were treated with sulfadoxine-pyrimethamine and followed up for 28 days. After losses to follow-up (n = 1) or elimination of DNA samples due to mixed parasite populations with pyrimethamine-sensitive and pyrimethamine-resistant profiles (n = 3), parasite genomic DNA from day 0 blood samples of six patients were analyzed by DNA sequencing. Three patients who were cured had isolates characterized by a wild-type or mutant dihydrofolate reductase gene (with one or two mutations) and a wild-type dihydropteroate synthase gene. Three other patients who failed to respond to sulfadoxine-pyrimethamine treatment carried isolates with triple dihydrofolate reductase gene mutations and either a wild-type or a mutant dihydropteroate synthase gene. Three dihydrofolate reductase gene codons (51, 59, and 108) may be reliable genetic markers that can accurately predict the clinical outcome of sulfadoxine-pyrimethamine treatment in Africa.

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