Isolation Of Insertion Mutants Research Articles

Genetic transformation, electrophoretic karyotyping and isolation of insertional mutants in the tree pathogenic fungus Neonectria galligena

SummaryThe ascomycete tree pathogen Neonectria galligena was transformed using several plasmid vectors containing the bacterial hygromycin phosphotransferase gene (hph) conferring resistance to hygromycin B. Protoplasts were produced by digesting germinating conidia in a Novozym 234 solution osmotically stabilized with NaCl. The protoplasts regenerated at ca. 70% on complete medium supplemented with either sucrose or NaCl. Up to 115 hygromycin B‐resistant transformants per microgram of transforming DNA per 108 protoplasts were obtained. Southern analysis of genomic DNA from the transformants, separated by either standard agarose gel electrophoresis or by pulsed field gel electrophoresis, confirmed that hygromycin B resistance resulted from integration of the vector. Transformants expressing the green fluorescent protein were also obtained. A total of 209 transformants were then screened for aggressiveness and mycelial radial growth. Four mutants were identified including one mutant with reduced aggressiveness but normal growth in vitro, two mutants with reduced growth in vitro but normal aggressiveness, and one mutant in which both aggressiveness and in vitro growth were reduced.

Arabidopsis genes essential for seedling viability: isolation of insertional mutants and molecular cloning.

We have undertaken a large-scale genetic screen to identify genes with a seedling-lethal mutant phenotype. From screening approximately 38,000 insertional mutant lines, we identified >500 seedling-lethal mutants, completed cosegregation analysis of the insertion and the lethal phenotype for >200 mutants, molecularly characterized 54 mutants, and provided a detailed description for 22 of them. Most of the seedling-lethal mutants seem to affect chloroplast function because they display altered pigmentation and affect genes encoding proteins predicted to have chloroplast localization. Although a high level of functional redundancy in Arabidopsis might be expected because 65% of genes are members of gene families, we found that 41% of the essential genes found in this study are members of Arabidopsis gene families. In addition, we isolated several interesting classes of mutants and genes. We found three mutants in the recently discovered nonmevalonate isoprenoid biosynthetic pathway and mutants disrupting genes similar to Tic40 and tatC, which are likely to be involved in chloroplast protein translocation. Finally, we directly compared T-DNA and Ac/Ds transposon mutagenesis methods in Arabidopsis on a genome scale. In each population, we found only about one-third of the insertion mutations cosegregated with a mutant phenotype.

Evidence that the RdeA protein is a component of a multistep phosphorelay modulating rate of development in Dictyostelium.

We have isolated an insertional mutant of Dictyostelium discoideum that aggregated rapidly and formed spores and stalk cells within 14 h of development instead of the normal 24 h. We have shown by parasexual genetics that the insertion is in the rdeA locus and have cloned the gene. It encodes a predicted 28 kDa protein (RdeA) that is enriched in charged residues and is very hydrophilic. Constructs with the DNA for the c-Myc epitope or for the green fluorescent protein indicate that RdeA is not compartmentalized. RdeA displays homology around a histidine residue at amino acid 65 with members of the H2 module family of phosphotransferases that participate in multistep phosphoryl relays. Replacement of this histidine rendered the protein inactive. The mutant is complemented by transformation with the Ypd1 gene of Saccharomyces cerevisiae, itself an H2 module protein. We propose that RdeA is part of a multistep phosphorelay system that modulates the rate of development.

Nucleotide sequence of Streptococcus mutans superoxide dismutase gene and isolation of insertion mutants.

A gene (sod) encoding superoxide dismutase (SOD) was cloned from Streptococcus mutans in Escherichia coli, and its nucleotide sequence was determined. The presumptive amino acid sequence of its product revealed that the SOD is basically of Mn type. Insertional inactivation of the sod gene resulted in the loss of SOD activity in crude extracts, indicating that the gene represents the only functional gene for SOD in S. mutans. Moreover, Southern blot analysis indicated that the S. mutans chromosome had no additional gene which was hybridizable with an oligonucleotide probe specific for an SOD motif. The SOD-deficient mutants were able to grow aerobically, albeit more slowly than the parent strains.