RESUMEN
Gene targeting is a commonly used method to reveal the function of genes. Although it is an attractive tool for molecular studies, it can frequently be a challenge because its efficiency can be low and it requires the screening of a large number of transformants. Generally, these problems originate from the elevated level of ectopic integration caused by non-homologous DNA end joining (NHEJ). To eliminate this problem, NHEJ-related genes are frequently deleted or disrupted. Although these manipulations can improve gene targeting, the phenotype of the mutant strains raised the question of whether mutations have side effects. The aim of this study was to disrupt the lig4 gene in the dimorphic fission yeast, S. japonicus, and investigate the phenotypic changes of the mutant strain. The mutant cells have shown various phenotypic changes, such as increased sporulation on complete medium, decreased hyphal growth, faster chronological aging, and higher sensitivity to heat shock, UV light, and caffeine. In addition, higher flocculation capacity has been observed, especially at lower sugar concentrations. These changes were supported by transcriptional profiling. Many genes belonging to metabolic and transport processes, cell division, or signaling had altered mRNA levels compared to the control strain. Although the disruption improved the gene targeting, we assume that the lig4 inactivation can cause unexpected physiological side effects, and we have to be very careful with the manipulations of the NHEJ-related genes. To reveal the exact mechanisms behind these changes, further investigations are required.
