Binding of general transcription factors TFIID and TFIIA to basal promoters is rate-limiting for transcriptional initiation of eukaryotic protein-coding genes. Consequently, activator proteins interacting with subunits of TFIID and/or TFIIA can drastically increase the rate of initiation events. Yeast transcriptional activator Ino2 interacts with several Taf subunits of TFIID...
pET expression plasmids are widely used for producing recombinant proteins in Escherichia coli. Selection and maintenance of cells harboring a pET plasmid are possible using either a Tn3.1-type genetic fragment (which encodes a ß-lactamase and confers resistance to ß-lactam antibiotics) or a Tn903.1-type genetic fragment (which encodes an aminoglycoside-3’-phosphotransferase and...
First marketed as RoundUp, glyphosate is history’s most popular herbicide because of its low acute toxicity to metazoans and broad-spectrum effectiveness across plant species. The development of glyphosate-resistant crops has led to increased glyphosate use and consequences from the use of glyphosate-based herbicides (GBH). Glyphosate has entered the food supply, spurred...
In Candida parapsilosis, homozygous disruption of the two genes encoding trehalase activity increased the susceptibility to Itraconazole compared with the isogenic parental strain. The fungicidal effect of this azole can largely be counteracted by preincubating growing cells with rotenone and the protonophore 2,4-Dinitrophenol. In turn, measurement of endogenous reactive oxygen...
In fungi, the cell wall plays a crucial role in morphogenesis and response to stress from the external environment. Chitin is one of the main cell wall components in many filamentous fungi. In Aspergillus nidulans, a class III chitin synthase ChsB plays a pivotal role in hyphal extension and morphogenesis. However, little is known about post-translational modifications of ChsB...
Understanding where proteins are localized in a bacterial cell is essential for understanding their function and regulation. This is particularly important for proteins that are involved in cell division, which localize at the division septum and assemble into highly regulated complexes. Current knowledge of these complexes has been greatly facilitated by super-resolution imaging...
Transcriptional corepressors Sin3, Cyc8 and Tup1 are important for downregulation of gene expression by recruiting various histone deacetylases once they gain access to defined genomic locations by interaction with pathway-specific repressor proteins. In this work we systematically investigated whether 17 yeast repressor proteins (Cti6, Dal80, Fkh1, Gal80, Mig1, Mot3, Nrg1, Opi1...
Fungal and plant mitochondria are known to exchange DNA with retroviral plasmids. Transfer of plasmid DNA to the organellar genome is best known and occurs through wholesale insertion of the plasmid. Less well known is the transfer of organellar DNA to plasmids, in particular tRNA genes. Presently, it is unknown whether fungal plasmids can adopt mitochondrial functions such as...
An extensive mutational analysis of RPL33A, encoding the yeast ribosomal protein L33A (eL33) allowed us to identify several novel rpl33a mutants with different translational phenotypes. Most of the rpl33a mutants are defective in the processing of 35S and 27S pre-rRNA precursors and the production of mature rRNAs, exhibiting reductions in the amounts of ribosomal subunits and...
The multiprotein Fab1p/PIKfyve-complex regulating the abundance of the phospholipid phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2) is highly conserved among eukaryotes. In yeast/mammals, it is composed of the phosphatidylinositol 3-phosphate 5-kinase Fab1p/PIKfyve, the PtdIns(3,5)P2 phosphatase Fig4p/Sac3 and the scaffolding subunit Vac14p/ArPIKfyve. The complex is located...
Synthetic Biology is revolutionizing biological research by introducing principles of mechanical engineering, including the standardization of genetic parts and standardized part assembly routes. Both are realized in the Modular Cloning (MoClo) strategy. MoClo allows for the rapid and robust assembly of individual genes and multigene clusters, enabling iterative cycles of gene...
Treating yeast cells with the replication inhibitor hydroxyurea activates the S phase checkpoint kinase Rad53, eliciting responses that block DNA replication origin firing, stabilize replication forks, and prevent premature extension of the mitotic spindle. We previously found overproduction of Stn1, a subunit of the telomere-binding Cdc13–Stn1–Ten1 complex, circumvents Rad53...
Dbf4 is the cyclin-like subunit for the Dbf4-dependent protein kinase (DDK), required for activating the replicative helicase at DNA replication origin that fire during S phase. Dbf4 also functions as an adaptor, targeting the DDK to different groups of origins and substrates. Here we report a genome-wide analysis of origin firing in a budding yeast mutant, dbf4-zn, lacking the...
Vibrio parahaemolyticus is a waterborne pathogen that can cause acute gastroenteritis, wound infection, and septicemia in humans. The molecular basis of its pathogenicity is not yet fully understood. Phages are found most abundantly in aquatic environments and play a critical role in horizontal gene transfer. Nevertheless, current literature on biological roles of prophage...
Mono-methylation of the fourth lysine on the N-terminal tail of histone H3 was found to support the induction of RNA polymerase II transcription in S. cerevisiae during nutrient stress. In S. cerevisiae, the mono-, di- and tri-methylation of lysine 4 on histone H3 (H3K4) is catalyzed by the protein methyltransferase, Set1. The three distinct methyl marks on H3K4 act in discrete...
The role of general transcription factor TFIIB in transcription extends well beyond its evolutionarily conserved function in initiation. Chromatin localization studies demonstrating binding of TFIIB to both the 5’ and 3’ ends of genes in a diverse set of eukaryotes strongly suggested a rather unexpected role of the factor in termination. TFIIB indeed plays a role in termination...
Magic spot synthetases are emerging targets to overcome persistence caused by stringent response. The ‘stringent response’ is a bacterial stress survival mechanism, which results in the accumulation of alarmones (also called Magic spots) leading to the formation of dormant persister cells. These ‘sleeper cells’ evade antibiotic treatment and could result in relapse of infection...
The increase in multidrug-resistant pathogenic bacteria has become a problem worldwide. Currently there is a strong focus on the development of novel antimicrobials, including antimicrobial peptides (AMP) and antimicrobial antisense agents. While the majority of AMP have membrane activity and kill bacteria through membrane disruption, non-lytic AMP are non-membrane active...
Under non-inducing conditions (absence of galactose), yeast structural genes of the GAL regulon are repressed by Gal80, preventing interaction of Gal4 bound to UASGAL promoter motifs with general factors of the transcriptional machinery. In this work, we show that Gal80 is also able to interact with histone deacetylase-recruiting corepressor proteins Cyc8 and Tup1, indicating an...
As the limiting component of the budding yeast telomerase, the Tlc1 RNA must undergo multiple consecutive modifications and rigorous quality checks throughout its lifecycle. These steps will ensure that only correctly processed and matured molecules are assembled into telomerase complexes that subsequently act at telomeres. The complex pathway of Tlc1 RNA maturation, involving 5...
As the limiting component of the budding yeast telomerase, the Tlc1 RNA must undergo multiple consecutive modifications and rigorous quality checks throughout its lifecycle. These steps will ensure that only correctly processed and matured molecules are assembled into telomerase complexes that subsequently act at telomeres. The complex pathway of Tlc1 RNA maturation, involving 5...
Fungal pathogens, from phytopathogenic fungus to human pathogens, are able to alternate between the yeast-like form and filamentous forms. This morphological transition (dimorphism) is in close connection with their pathogenic lifestyles and with their responses to changing environmental conditions. The mechanisms governing these morphogenetic conversions are still not fully...