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A system of biological containment for recombinant DNA experiments in Saccharomyces cerevisiae (Brewer's/Baker's yeast) is described. The principle of containment is sterility: the haploid host strains all contain a mating-type-non-specific sterile mutation. The hosts also contain four auxotrophic mutations suitable for selection for the various kinds of(More)
We have increased the lysine content in the seeds of canola and soybean plants by circumventing the normal feedback regulation of two enzymes of the biosynthetic pathway, aspartokinase (AK) and dihydrodipicolinic acid synthase (DHDPS). Lysine-feedback-insensitive bacterial DHDPS and AK enzymes encoded by the Corynebacterium dapA gene and a mutant E. coli(More)
Targeted mutagenesis, editing of endogenous maize (Zea mays) genes, and site-specific insertion of a trait gene using clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas)-guide RNA technology are reported in maize. DNA vectors expressing maize codon-optimized Streptococcus pyogenes Cas9 endonuclease and single guide RNAs were(More)
Sulfometuron methyl (SM), a potent new sulfonylurea herbicide, inhibits growth of the yeast Saccharomyces cerevisiae on minimal media. Sixty-six spontaneous mutants resistant to SM were isolated. All of the resistance mutations segregate 2:2 in tetrads; 51 of the mutations are dominant, five are semidominant and ten are recessive. The mutations occur in(More)
Yeast DNA fragments that confer multiple drug resistance when amplified were isolated. Cells containing a yeast genomic library cloned in the high copy autonomously replicating vector, YEp24, were plated on medium containing cycloheximide. Five out of 100 cycloheximide-resistant colonies were cross-resistant to the unrelated inhibitor, sulfometuron methyl,(More)
Marker-gene-free transgenic soybean plants were produced by isolating a developmentally regulated embryo-specific gene promoter, app1, from Arabidopsis and developing a self-activating gene excision system using the P1 bacteriophage Cre/loxP recombination system. To accomplish this, the Cre recombinase gene was placed under control of the app1 promoter and,(More)
Recently discovered bacteria and archaea adaptive immune system consisting of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) endonuclease has been explored in targeted genome editing in different species. Streptococcus pyogenes Cas9-guide RNA (gRNA) was successfully applied to generate targeted mutagenesis,(More)
The liguleless locus (liguleless1) was chosen for demonstration of targeted mutagenesis in maize using an engineered endonuclease derived from the I-CreI homing endonuclease. A single-chain endonuclease, comprising a pair of I-CreI monomers fused into a single polypeptide, was designed to recognize a target sequence adjacent to the LIGULELESS1 (LG1) gene(More)
We have determined the nucleotide sequence of the yeast ILV2 gene which codes for the amino acid biosynthetic enzyme acetolactate synthase (ALS). ALS has recently been shown to be the target in bacteria, yeast and plants, of the potent new herbicide sulfometuron methyl. The coding sequence for the ILV2 polypeptide contains 2061 base pairs. Comparison of(More)
We obtained strains of yeast with large segments of 2 mu plasmid DNA integrated at several chromosomal locations by selecting genetically for recombination between a chromosomal sequence carried on a 2 mu-circle-containing hybrid plasmid and a homologous sequence on the chromosome. In all diploids examined, the presence of 2 mu circle sequences causes a(More)