Development of a fungal transformation system based on selection of sequences with promoter activity

1987 ◽  
Vol 7 (9) ◽  
pp. 3297-3305
Author(s):  
B G Turgeon ◽  
R C Garber ◽  
O C Yoder
Keyword(s):  
Genomic Dna ◽  
Pathogenic Fungus ◽  
Low Frequency ◽  
Hygromycin B ◽  
Fungal Transformation ◽  
Dna Fragments ◽  
Transformation System ◽  
Chromosomal Dna ◽  
General Utility ◽  
Novel Strategy

A novel strategy was used to develop a transformation system for the plant pathogenic fungus Cochliobolus heterostrophus. Sequences capable of driving the expression of a gene conferring resistance to the antibiotic hygromycin B in C. heterostrophus were selected from a library of genomic DNA fragments and used, with the selectable marker, as the basis for transformation. The library of random 0.5- to 2.0-kilobase-pair fragments of C. heterostrophus genomic DNA was inserted at the 5' end of a truncated, promoterless Escherichia coli hygromycin B phosphotransferase gene (hygB) whose product confers resistance to hygromycin B. C. heterostrophus protoplasts were transformed with the library and selected for resistance. Resistant colonies arose at low frequency. Each colony contained a transformation vector stably integrated into chromosomal DNA. When the transforming DNA was recovered from the genome and introduced into C. heterostrophus, resistant colonies appeared at higher frequency. We determined the sequences of two of the C. heterostrophus DNA fragments which had been inserted at the 5' end of hygB in the promoter library and found that both made translational fusions with hygB. One of the two fusions apparently adds 65 and the other at least 86 amino acids to the N-terminus of the hygB product. Plasmids containing hygB-C. heterostrophus promoter fusions can be used unaltered to drive hygB expression in several other filamentous ascomycetes. This approach to achieving transformation may have general utility, especially for organisms with relatively undeveloped genetics.

scholarly journals Development of a fungal transformation system based on selection of sequences with promoter activity.

1987 ◽  
Vol 7 (9) ◽  
pp. 3297-3305 ◽  
Author(s):  
B G Turgeon ◽  
R C Garber ◽  
O C Yoder
Keyword(s):  
Genomic Dna ◽  
Pathogenic Fungus ◽  
Low Frequency ◽  
Hygromycin B ◽  
Fungal Transformation ◽  
Dna Fragments ◽  
Transformation System ◽  
Chromosomal Dna ◽  
General Utility ◽  
Novel Strategy

A novel strategy was used to develop a transformation system for the plant pathogenic fungus Cochliobolus heterostrophus. Sequences capable of driving the expression of a gene conferring resistance to the antibiotic hygromycin B in C. heterostrophus were selected from a library of genomic DNA fragments and used, with the selectable marker, as the basis for transformation. The library of random 0.5- to 2.0-kilobase-pair fragments of C. heterostrophus genomic DNA was inserted at the 5' end of a truncated, promoterless Escherichia coli hygromycin B phosphotransferase gene (hygB) whose product confers resistance to hygromycin B. C. heterostrophus protoplasts were transformed with the library and selected for resistance. Resistant colonies arose at low frequency. Each colony contained a transformation vector stably integrated into chromosomal DNA. When the transforming DNA was recovered from the genome and introduced into C. heterostrophus, resistant colonies appeared at higher frequency. We determined the sequences of two of the C. heterostrophus DNA fragments which had been inserted at the 5' end of hygB in the promoter library and found that both made translational fusions with hygB. One of the two fusions apparently adds 65 and the other at least 86 amino acids to the N-terminus of the hygB product. Plasmids containing hygB-C. heterostrophus promoter fusions can be used unaltered to drive hygB expression in several other filamentous ascomycetes. This approach to achieving transformation may have general utility, especially for organisms with relatively undeveloped genetics.

Genetic transformation of the vascular wilt fungusVerticillium dahliae

1995 ◽  
Vol 73 (5) ◽  
pp. 710-715 ◽  
Author(s):  
Katherine F. Dobinson
Keyword(s):  
Dna Sequences ◽  
Chromosome Rearrangement ◽  
Electrophoretic Karyotype ◽  
Hygromycin B ◽  
Fungal Transformation ◽  
Vascular Wilt ◽  
Transformation System ◽  
Multiple Copies ◽  
Vector Dna

To facilitate genetic analysis of pathogenicity of Verticillium dahliae, a vascular wilt pathogen, a DNA-mediated transformation system has been developed. Resistance to hygromycin B was obtained by transforming spheroplasts with the cosmid vector pAN7-2. Transformation efficiencies ranged between 3 and 5 transformants/μg vector DNA. The transforming DNA was integrated into the V. dahliae genome, in single and multiple copies and in tandem array. In several multicopy transformants, minor alterations in the integrated DNA sequences were evident following extensive vegetative growth in the absence of hygromycin B. Electrophoretic karyotype analysis also provided direct evidence of chromosome rearrangements in two transformants. The availability of a transformation system for V. dahliae will facilitate the cloning and characterization of genes that are important for pathogenicity and development. Key words: Verticillium wilt, fungal transformation, electrophoretic karyotype, hygromycin B resistance, chromosome rearrangement.

Enzymatic amplification and characterization of large DNA fragments from genomic DNA

Gene ◽  
1988 ◽  
Vol 71 (1) ◽  
pp. 211-216 ◽  
Author(s):  
Phouthone Keohavong ◽  
Cindy C. Wang ◽  
Rita S. Cha ◽  
William G. Thilly
Keyword(s):  
Genomic Dna ◽  
Dna Fragments ◽  
Enzymatic Amplification

Effects of mutation position on frequency of marker rescue by homologous recombination

1992 ◽  
Vol 12 (8) ◽  
pp. 3609-3613
Author(s):  
L Jiang ◽  
A Connor ◽  
M J Shulman
Keyword(s):  
Homologous Recombination ◽  
Normal Function ◽  
Constant Region ◽  
Dna Fragments ◽  
Wild Type ◽  
End Effect ◽  
Chromosomal Dna ◽  
Base Pairs ◽  
Marker Rescue ◽  
Immunoglobulin Mu

Homologous recombination between transferred and chromosomal DNA can be used for mapping mutations by marker rescue, i.e., by identifying which segment of wild-type DNA can recombine with the mutant chromosomal gene and restore normal function. In order to define how much the fragments should overlap each other for reliable mapping, we have measured how the frequency of marker rescue is affected by the position of the chromosomal mutation relative to the ends of the transferred DNA fragments. For this purpose, we used several DNA fragments to effect marker rescue in two mutant hybridomas which bear mutations 673 bp apart in the exons encoding the second and third constant region domains of the immunoglobulin mu heavy chain. The frequency of marker rescue decreased greatly when the mutation was located near one of the ends of the fragments, the results indicating that fragments should be designed to overlap by at least several hundred base pairs. Possible explanations for this "end effect" are considered.

Preparation of a "functional library" of African green monkey DNA fragments which substitute for the processing/polyadenylation signal in the herpes simplex virus type 1 thymidine kinase gene

1983 ◽  
Vol 3 (4) ◽  
pp. 643-653
Author(s):  
G M Santangelo ◽  
C N Cole
Keyword(s):  
Gene Expression ◽  
Thymidine Kinase ◽  
Low Frequency ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
African Green Monkey ◽  
Dna Fragments ◽  
Coding Region ◽  
Kinase Gene ◽  
Green Monkey

Fragments of African green monkey (Cercopithecus aethiops) DNA (3.5 to 18.0 kilobases) were inserted downstream from the thymidine kinase (TK, tk) coding region in pTK206/SV010, a gene construct which lacks both copies of the hexanucleotide 5'-AATAAA-3' and contains a simian virus 40 origin of replication, allowing it to replicate in Cos-1 cells. No polyadenylated tk mRNA was detected in Cos-1 cells transfected by pTK206/SV010. The ability of simian DNA fragments to restore tk gene expression was examined by measuring the incorporation of [125I]iododeoxycytidine into DNA in Cos-1 cells transfected by pTK206/SV010 insertion derivatives. tk gene expression was restored by the insertion in 56 of the 67 plasmids analyzed, and the level of expression equaled or exceeded that obtained with the wild-type tk gene in 30 of these. In all plasmids examined that showed restoration of tk gene expression, polyadenylated tk mRNA of discrete size was detected. The sizes of these tk mRNAs were consistent with the existence of processing and polyadenylation signals within the inserted DNA fragments. The frequency with which inserted fragments restored tk gene expression suggests that the minimal signal for processing and polyadenylation is a hexanucleotide (AAUAAA or a similar sequence). LTK- cells were biochemically transformed to TK+ with representative insertion constructs. pTK206/SV010 transformed LTK- cells at a very low frequency; the frequency of transformation with insertion derivatives was 40 to 12,000 times higher.

Identification of developmental regulatory genes in Aspergillus nidulans by overexpression.

Genetics ◽  
1995 ◽  
Vol 139 (2) ◽  
pp. 537-547 ◽  
Author(s):  
J F Marhoul ◽  
T H Adams
Keyword(s):  
Growth Inhibition ◽  
Aspergillus Nidulans ◽  
Genomic Dna ◽  
Regulatory Gene ◽  
Regulatory Genes ◽  
Dna Fragments ◽  
Expression Library ◽  
Phenotypic Changes ◽  
Inhibition Of Growth ◽  
Induction Sequence

Abstract Overexpression of several Aspergillus nidulans developmental regulatory genes has been shown to cause growth inhibition and development at inappropriate times. We set out to identify previously unknown developmental regulators by constructing a nutritionally inducible A. nidulans expression library containing small, random genomic DNA fragments inserted next to the alcA promoter [alcA(p)] in an A. nidulans transformation vector. Among 20,000 transformants containing random alcA(p) genomic DNA fusion constructs, we identified 66 distinct mutant strains in which alcA(p) induction resulted in growth inhibition as well as causing other detectable phenotypic changes. These growth inhibited mutants were divided into 52 FIG (Forced expression Inhibition of Growth) and 14 FAB (Forced expression Activation of brlA) mutants based on whether or not alcA(p) induction resulted in accumulation of mRNA for the developmental regulatory gene brlA. In four FAB mutants, alcA(p) induction not only activated brlA expression but also caused hyphae to differentiate into reduced conidiophores that produced viable spores from the tips as is observed after alcA(p)::brlA induction. Sequence analyses of the DNA fragments under alcA(p) control in three of these four sporulating strains showed that in two cases developmental activation resulted from overexpression of previously uncharacterized genes, whereas in the third strain, the alcA(p) was fused to brlA. The potential uses for this strategy in identifying genes whose overexpression results in specific phenotypic changes like developmental induction are discussed.

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