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.

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.

scholarly journals Stoichiometry and compositional plasticity of the yeast nuclear pore complex revealed by quantitative fluorescence microscopy

2018 ◽  
Vol 115 (17) ◽  
pp. E3969-E3977 ◽  
Author(s):  
Sasikumar Rajoo ◽  
Pascal Vallotton ◽  
Evgeny Onischenko ◽  
Karsten Weis
Keyword(s):  
Nuclear Pore Complex ◽  
Yeast Cells ◽  
Nuclear Pore ◽  
Altered Expression ◽  
Copy Numbers ◽  
Quantitative Image ◽  
Multiple Copies ◽  
Almost All ◽  
High Degree

The nuclear pore complex (NPC) is an eightfold symmetrical channel providing selective transport of biomolecules across the nuclear envelope. Each NPC consists of ∼30 different nuclear pore proteins (Nups) all present in multiple copies per NPC. Significant progress has recently been made in the characterization of the vertebrate NPC structure. However, because of the estimated size differences between the vertebrate and yeast NPC, it has been unclear whether the NPC architecture is conserved between species. Here, we have developed a quantitative image analysis pipeline, termed nuclear rim intensity measurement (NuRIM), to precisely determine copy numbers for almost all Nups within native NPCs of budding yeast cells. Our analysis demonstrates that the majority of yeast Nups are present at most in 16 copies per NPC. This reveals a dramatic difference to the stoichiometry determined for the human NPC, suggesting that despite a high degree of individual Nup conservation, the yeast and human NPC architecture is significantly different. Furthermore, using NuRIM, we examined the effects of mutations on NPC stoichiometry. We demonstrate for two paralog pairs of key scaffold Nups, Nup170/Nup157 and Nup192/Nup188, that their altered expression leads to significant changes in the NPC stoichiometry inducing either voids in the NPC structure or substitution of one paralog by the other. Thus, our results not only provide accurate stoichiometry information for the intact yeast NPC but also reveal an intriguing compositional plasticity of the NPC architecture, which may explain how differences in NPC composition could arise in the course of evolution.

Cloning and characterization of DNA sequences amplified in multidrug-resistant Djungarian hamster and mouse cells

1987 ◽  
Vol 13 (6) ◽  
pp. 609-619 ◽  
Author(s):  
A. V. Gudkov ◽  
O. B. Chernova ◽  
A. R. Kazarov ◽  
B. P. Kopnin
Keyword(s):  
Dna Sequences ◽  
Multidrug Resistant ◽  
Djungarian Hamster ◽  
Mouse Cells

Molecular cloning and characterization of an activated human c-raf-1 gene

1987 ◽  
Vol 7 (5) ◽  
pp. 1776-1781
Author(s):  
M Fukui ◽  
T Yamamoto ◽  
S Kawai ◽  
F Mitsunobu ◽  
K Toyoshima
Keyword(s):  
Dna Sequences ◽  
Promoter Sequence ◽  
Small Population ◽  
Dna Transfection ◽  
Amino Terminal ◽  
Human Dna ◽  
Nih 3T3 ◽  
Amino Terminal Sequence ◽  
Tumor Dna

Results of previous studies have shown that a raf-related transforming DNA sequence is present in NIH 3T3 transformants that are derived from GL-5-JCK human glioblastoma DNA transfection. The transforming DNA was molecularly cloned by using cosmid vector pJB8 to determine its structure and origin. Analyses of selected clones revealed that the transforming DNA consisted of three portions of human DNA sequences, with the 3' half of the c-raf-1 gene as its middle portion. This raf region was about 20 kilobases long and contained exons 8 to 17 and the poly(A) addition site. RNA blot analysis showed that the raf-related transforming DNA was transcribed into 5.3-, 4.8-, and 2.5-kilobase mRNAs; the 2.5-kilobase transcript was thought to be the major transcript. Immunoprecipitation analyses revealed that a 44-kilodalton raf-related protein was specifically expressed in the NIH 3T3 transformants. The raf-related transforming DNA was considered to be activated when its amino-terminal sequence was truncated and the DNA was coupled with a foreign promoter sequence. On hybridization analysis of the original GL-5-JCK glioblastoma DNA, no rearrangement of c-raf-1 was detectable in the tumor DNA. The rearrangement of c-raf-1 may have occurred during transfection or may have been present in a small population of the original tumor cells as a result of tumor progression.

Identification and characterization of the centromere from chromosome XIV in Saccharomyces cerevisiae

1985 ◽  
Vol 5 (11) ◽  
pp. 2887-2893
Author(s):  
M Neitz ◽  
J Carbon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Restriction Fragment ◽  
Dna Sequences ◽  
Yeast Genome ◽  
Ura3 Gene ◽  
Dna Restriction ◽  
Meiotic Analyses ◽  
The Right ◽  
In Cis

A functional centromere located on a small DNA restriction fragment from Saccharomyces cerevisiae was identified as CEN14 by integrating centromere-adjacent DNA plus the URA3 gene by homologous recombination into the yeast genome and then by localizing the URA3 gene to chromosome XIV by standard tetrad analysis. DNA sequence analysis revealed that CEN14 possesses sequences (elements I, II, and III) that are characteristic of other yeast centromeres. Mitotic and meiotic analyses indicated that the CEN14 function resides on a 259-base-pair (bp) RsaI-EcoRV restriction fragment, containing sequences that extend only 27 bp to the right of the element I to III region. In conjunction with previous findings on CEN3 and CEN11, these results indicate that the specific DNA sequences required in cis for yeast centromere function are contained within a region about 150 bp in length.

Isolation of a yeast gene involved in species-specific pre-tRNA processing

1988 ◽  
Vol 8 (12) ◽  
pp. 5140-5149
Author(s):  
S S Wang ◽  
A K Hopper
Keyword(s):  
Dna Sequences ◽  
Genomic Library ◽  
Nonsense Suppression ◽  
Suppressor Activity ◽  
Specific Product ◽  
Trna Processing ◽  
Trna Splicing ◽  
Trna Species ◽  
Multiple Copies ◽  
Species Specific

To identify genes involved in pre-tRNA processing, we searched for yeast DNA sequences that specifically enhanced the expression of the SUP4(G37) gene. The SUP4(G37) gene possesses a point mutation at position 37 of suppressor tRNA(Tyr). This lesion results in a reduced rate of pre-tRNA splicing and a decreased level of nonsense suppression. A SUP4(G37) strain was transformed with a yeast genomic library, and the transformants were screened for increased suppressor activity. One transformant contained a plasmid that encoded an unessential gene, STP1, that in multiple copies enhanced the suppression of SUP4(G37) and caused increased production of mature SUP4(G37) product. Disruption of the genomic copy of STP1 resulted in a reduced efficiency of SUP4-mediated suppression and the accumulation of pre-tRNAs. Not all intron-containing pre-tRNAs were affected by the stp1-disruption. At least five of the nine families of pre-tRNAs were affected. Two other species, pre-tRNA(Ile) and pre-tRNA(3Leu), were not. We propose that STP1 encodes a tRNA species-specific product that functions as a helper for pre-tRNA splicing. The STP1 product may interact with pre-tRNAs to generate a structure that is efficiently recognized by splicing machinery.

scholarly journals Karyotype Characterization of Nine Periwinkle Species (Gastropoda, Littorinidae)

Genes ◽  
2018 ◽  
Vol 9 (11) ◽  
pp. 517 ◽  
Author(s):  
Daniel García-Souto ◽  
Sandra Alonso-Rubido ◽  
Diana Costa ◽  
José Eirín-López ◽  
Emilio Rolán-Álvarez ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Chromosome Numbers ◽  
Gene Clusters ◽  
Chromosomal Mapping ◽  
Closely Related Species ◽  
Submetacentric Chromosome ◽  
The Family ◽  
Littorina Arcana

Periwinkles of the family Littorinidae (Children, 1834) are common members of seashore littoral communities worldwide. Although the family is composed of more than 200 species belonging to 18 genera, chromosome numbers have been described in only eleven of them. A molecular cytogenetic analysis of nine periwinkle species, the rough periwinkles Littorina arcana, L. saxatilis, and L. compressa, the flat periwinkles L. obtusata and L. fabalis, the common periwinkle L. littorea, the mangrove periwinkle Littoraria angulifera, the beaded periwinkle Cenchritis muricatus, and the small periwinkle Melarhaphe neritoides was performed. All species showed diploid chromosome numbers of 2n = 34, and karyotypes were mostly composed of metacentric and submetacentric chromosome pairs. None of the periwinkle species showed chromosomal differences between male and female specimens. The chromosomal mapping of major and minor rDNA and H3 histone gene clusters by fluorescent in situ hybridization demonstrated that the patterns of distribution of these DNA sequences were conserved among closely related species and differed among less related ones. All signals occupied separated loci on different chromosome pairs without any evidence of co-localization in any of the species.

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