ABSTRACT
One of the most productive strategies for finding the functions of proteins is to study the consequences of loss of protein function. For this purpose, cells or organisms with a knockout of the gene encoding the protein of interest are obtained. However, many proteins perform important functions and cells or organisms could suddenly lose fitness when the function of a protein is lost. For such proteins, the most productive strategy is to use inducible protein degradation systems. A system of auxin-dependent protein degradation is often implemented. To use this system, it is sufficient to introduce a transgene encoding a plant-derived auxin-dependent ubiquitin ligase into mammalian cells and insert a sequence encoding a degron domain into the gene of interest. A crucial aspect of development of cell lines engineered for inducible protein depletion is the selection of cell clones with efficient auxin-dependent degradation of the protein of interest. To select clones induced by depletion of the architectural chromatin proteins RAD21 (a component of the cohesin complex) and SMC2 (a component of the condensin complex), we propose to use the morphology of metaphase chromosomes as a convenient functional test. In this work, we obtained a series of clones of human HAP1 cells carrying the necessary genetic constructs for inducible depletion of RAD21 and SMC2. The degradation efficiency of the protein of interest was assessed by flow cytometry, Western blotting and metaphase chromosome morphology test. Based on our tests, we showed that the clones we established with the SMC2 degron effectively and completely lose protein function when induced by auxin. However, none of the HAP1 clones we created with the RAD21 degron showed complete loss of RAD21 function upon induction of degradation by auxin. In addition, some clones showed evidence of loss of RAD21 function even in the absence of induction. The chromosome morphology test turned out to be a convenient and informative method for clone selection. The results of this test are in good agreement with flow cytometry analysis and Western blotting data.
ABSTRACT
Many plants and animals have symbiotic relationships with microorganisms, including bacteria. The interactions between bacteria and their hosts result in different outcomes for the host organism. The outcome can be neutral, harmful or have beneficial effects for participants. Remarkably, these relationships are not static, as they change throughout an organism's lifetime and on an evolutionary scale. One of the structures responsible for relationships in bacteria is O-antigen. Depending on the characteristics of its components, the bacteria can avoid the host's immune response or establish a mutualistic relationship with it. O-antigen is a key component in Gram-negative bacteria's outer membrane. This component facilitates interaction between the bacteria and host immune system or phages. The variability of the physical structure is caused by the genomic variability of genes encoding O-antigen synthesis components. The genes and pathways of O-polysaccharide (OPS) synthesis were intensively investigated mostly for Enterobacteriaceae species. Considering high genetic and molecular diversity of this structure even between strains, these findings may not have caught the entire variety possibly presented in non-model species. The current study presents a comparative analysis of genes associated with O-antigen synthesis in bacteria of the Oxalobacteraceae family. In contrast to existing studies based on PCR methods, we use a bioinformatics approach and compare O- antigens at the level of clusters rather than individual genes. We found that the O-antigen genes of these bacteria are represented by several clusters located at a distance from each other. The greatest similarity of the clusters is observed within individual bacterial genera, which is explained by the high variability of O-antigens. The study describes similarities of OPS genes inherent to the family as a whole and also considers individual unique cases of O-antigen genetic variability inherent to individual bacteria.
