In the budding yeast Saccharomyces cerevisiae, the cell division cycle and sporulation are mutually exclusive cell fates; glucose, which stimulates the cell division cycle, is a potent inhibitor of sporulation. Addition of moderate concentrations of glucose (0.5%) to sporulation medium did not inhibit transcription of two key activators of sporulation, IME1 and IME2, but did increase levels of Sic1p, a cyclin-dependent kinase inhibitor, resulting in a block to meiotic DNA replication. The effects of glucose on Sic1p levels and DNA replication required Grr1p, a component of the SCF Grr1p ubiquitin ligase. Sic1p is negatively regulated by Ime2p kinase, and several observations indicate that glucose inhibits meiotic DNA replication through SCF Grr1p -mediated destruction of this kinase. First, Ime2p was destabilized in the presence of glucose, and this turnover required Grr1p, a second component of SCF Grr1p , Cdc53p, and an SCF Grr1p -associated E2 enzyme, Cdc34p. Second, Ime2p-ubiquitin conjugates were detected under conditions of rapid Ime2p turnover, and conjugation of Ime2p to ubiquitin required GRR1. Third, a mutant form of Ime2p (Ime2 ⌬PEST ), in which a putative Grr1p-interacting sequence was deleted, was more stable than wild-type Ime2p. Finally, expression of the IME2⌬PEST allele bypassed the block to meiotic DNA replication caused by 0.5% glucose. In addition, Grr1p is required for later events in sporulation independently of its role in Ime2p turnover.
Targeted gene replacement (TGR) using fragments generated by PCR is a widely-used technique for deleting genes in Saccharomyces cerevisiae. We found that the efficiency of this procedure, defined as the fraction of transformants that delete the targeted gene, varied by >10-fold depending on the sequence being targeted. We examined the effect of chromosomal position, length of homology and GC content on TGR efficiency. When URA3 was positioned at five different chromosomal locations, the efficiency of replacing this gene with LEU2 remained the same. Similarly, varying the length of homology from 35 to 60 bp had only a small effect on the efficiency of targeting (<50%), though an increase in the length of homology to 200 bp on one end of the disruption fragment did increase TGR efficiency. Strikingly, as GC content in the target sequence increased, the efficiency of targeting also increased. When TGR efficiency was high, the frequency of untargeted integration events was low. These results suggest two strategies for designing TGR primers: (i) use 40 bp targeting sequences containing 40-50% GC, and (ii) if necessary, increase TGR efficiency by extending the length of homology on one end of the disruption fragment.
We describe three extensions of the method of site-specific genomic (SSG) mutagenesis. These three extensions of SSG mutagenesis were used to generate precise insertion, deletion, and allele substitution mutations in the genome of the budding yeast, Saccharomyces cerevisiae. These mutations are termed precise because no attached sequences (e.g., marker genes or recombination sites) are retained once the method is complete. Because the method is PCR-based, neither DNA cloning nor synthesis of long oligonucleotides is required. We demonstrated the efficacy of these methods by deleting an ORF, inserting the tandem affinity purification (TAP) tag, and replacing a wild-type allele with a mutant allele.
Background: A valuable weapon in the arsenal available to yeast geneticists is the ability to introduce specific mutations into yeast genome. In particular, methods have been developed to introduce deletions into the yeast genome using PCR fragments. These methods are highly efficient because they do not require cloning in plasmids.
Screening for loose smut resistance in wheat is difficult. Selecting lines with DNA markers linked to loose smut resistance would be more reliable and less costly. Molecular markers linked to a race T10 loose smut resistance gene were identified using a F6 single seed descent segregating population. A RAPD marker and a RFLP marker were located on opposite flanks of the resistance gene and were shown to be loosely linked. The RAPD marker was converted to a user friendly polymorphic SCAR marker that represented a single genetically defined locus in hexaploid wheat. Using these two bracketing markers simultaneously, the error rate for T10 resistance selection due to crossing-over was reduced to 4%. These markers can be used for a faster and more reliable selection of T10 resistant plants than previous conventional loose smut ratings.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.