Unilateral ends-out gene targeting increases mistargeting through supporting extensive single-strand assimilation.

Ends-out gene targeting enables the swapping of endogenous alleles with exogenous ones through homologous recombination which bears great implications both fundamental and applicable. To address the recombination mechanism(s) behind it, an experimental system was designed to distinguish between a possible (but rarely active) unilateral and the expected bilateral targeting in the yeast Saccharomyces cerevisiae in which the proportions of the two alternative genetic outcomes are conceived to mirror the probabilities of the two scenarios. The quantitative analysis showed that the bilateral targeting was expectedly predominant. However, an analogous comparative analysis on a different experimental set suggested a prevalence of unilateral targeting unveiling an uncertainty whether the extensively resected targeting modules only mimic unilateral invasion. Based on this, a comprehensive qualitative analysis was conducted revealing a single basic ends-out gene targeting mechanism composed of two intertwined pathways differing in the way how the homologous invasion is initiated and/or the production of the intermediates is conducted. This study suggests that bilateral targeting lowers mistargeting plausibly by limiting strand assimilation, unlike unilateral targeting which may initiate extensive strand assimilation producing intermediates capable of supporting multiple genetic outcomes which leads to mistargeting. Some of these outcomes can also be produced by mimicking unilateral invasion.

Visualizing genomic data: The mixing perspective.

We report on a novel way to visualize genomic data. By considering genome coding sequences, cds, as sets of the N=61 non-stop codons, one obtains a partition of the total number of codons in each cds. Partitions exhibit a statistical property known as mixing character which characterizes how mixed the partition is. Mixing characters have been shown mathematically to exhibit a partial order known as majorization (Ruch, 1975). In previous work (Seitz and Kirwan, 2022) we developed an approach that combined mixing and entropy that is visualized as a scatter plot. If we consider all 1,121,505 partitions of 61 codons, this produces a plot we call the theoretical mixing space, TGMS. A normalization procedure is developed here and applied to real genomic data to produce the genome mixing signature, GMS. Example GMS's of 19 species, including Homo sapiens, are shown and discussed.

Insertion orientation within the cassette affects gene-targeting success during ends-out recombination in the yeast Saccharomyces cerevisiae.

Gene-targeting is one of the most important molecular tools for genomic manipulations for research and industrial purposes. However, many factors influence targeting fidelity undermining the efforts for accurate, fast, and reliable construction of genetically modified yeast strains. Therefore, it is of great academic interest that we uncover as many as possible parameters affecting the recombination mechanisms that enable targeting. Since usually, researchers choose the orientation of the insertion (marker) within the module at random, it seemed interesting to see whether the same module will achieve essentially the same targeting efficiency when the same marker was oriented alternatively concerning the same target gene. Thus, two loci (URA3 and LEU2) and one allele (ura3-52) in a haploid yeast genetic background were targeted by artificial modules bearing homologous insertions in alternative orientations being flanked by long asymmetrical targeting homology to either replace or disrupt a genomic target. Results showed that insertion orientation within the targeting module strongly influences targeting in yeast, regardless of the targeting approach.

Ecologically driven competence for exogenous DNA uptake in yeast.

Unlike prokaryotes, eukaryotic organisms do not seem to be equipped with natural cell process(es) designated for exogenous DNA uptake. However, it is barely known that under laboratory circumstances resembling wild fungal environment(s), at least some lower eukaryotes could become naturally competent for exogenous DNA uptake. Thus, apart from the known fact that non-manipulated cells of yeast Saccharomyces cerevisiae take exogenous DNA by conjugation with certain bacteria, there are also mechanical and physiological mechanisms enabling their transformation under environmental conditions. This clearly shows that lower eukaryotes are amenable to transformation without applying man-made technology (i.e., naturally). However, this topic failed to raise critical scientific interest. Therefore, this review aims to scrutinize the overall implication of the phenomenon stressing its fundamental and applicable importance. It also summarizes all axiomatic laboratory circumstances/vehicles hitherto known to provoke yeast competence naturally and critically discusses plausible mechanisms behind. Possible pathways underlying the phenomenon are emphasized and a unifying model is proposed. This story potentially spans several different research fields, from evolutionary genetics to genetic transformation technology.

Yeast competence for exogenous DNA uptake: towards understanding its genetic component.

The demonstration of spontaneous yeast competence shows that artificial transformation rests on naturally occurring cellular processes. Such natural competence is either biologically mediated or environmentally induced. For instance, wild yeast might be transformed through conjugation by cell-to-cell contact mediated either by Escherichia coli or Agrobacterium tumefaciens. Moreover, natural competence can be enhanced by mechanical and physiological mechanisms. On the other hand, artificial yeast competence is usually achieved by biological, chemical and physical manipulations. These eliminate or weaken natural obstacles blocking the way of the transforming DNA in order to mitigate its entrance into the cell (biological and chemical approach) or simply to bridge it by either electrical or biolistic force (physical). Thus yeast competence is controlled by intrinsic (genetic) and external (environmental) factors. Both intrinsic and external parameters affecting yeast competence were scrutinized leading to the identification of genes and biological processes participating in the phenomenon. Therefore natural yeast competence is a complex, quantitative genetic trait which may have allowed yeast to better adapt over evolutionary times.