Kinetic analysis of synaptonemal complex dynamics during meiosis of yeast Saccharomyces cerevisiae reveals biphasic growth and abortive disassembly.

The synaptonemal complex (SC) is a dynamic structure formed between chromosomes during meiosis which stabilizes and supports many essential meiotic processes such as pairing and recombination. In budding yeast, Zip1 is a functionally conserved element of the SC that is important for synapsis. Here, we directly measure the kinetics of Zip1-GFP assembly and disassembly in live cells of the yeast S. cerevisiae. The imaging of SC assembly in yeast is challenging due to the large number of chromosomes packed into a small nucleus. We employ a zip3Δ mutant in which only a few chromosomes undergo synapsis at any given time, initiating from a single site on each chromosome, thus allowing the assembly and disassembly kinetics of single SCs to be accurately monitored in living cells. SC assembly occurs with both monophasic and biphasic kinetics, in contrast to the strictly monophasic assembly seen in C. elegans. In wild-type cells, once maximal synapsis is achieved, programmed final disassembly rapidly follows, as Zip1 protein is actively degraded. In zip3Δ, this period is extended and final disassembly is prolonged. Besides final disassembly, we found novel disassembly events involving mostly short SCs that disappeared in advance of programmed final disassembly, which we termed "abortive disassembly." Abortive disassembly is distinct from final disassembly in that it occurs when Zip1 protein levels are still high, and exhibits a much slower rate of disassembly, suggesting a different mechanism for removal in the two types of disassembly. We speculate that abortive disassembly events represent defective or stalled SCs, possibly representing SC formation between non-homologs, that is then targeted for dissolution. These results reveal novel aspects of SC assembly and disassembly, potentially providing evidence of additional regulatory pathways controlling not just the assembly, but also the disassembly, of this complex cellular structure.

In Vivo Imaging of Budding Yeast Meiosis.

Tracking biological events in living cells provides kinetic information about biological processes that can be missed in more traditional methods using fixed samples at designated time intervals. Here we describe a methodology for in vivo fluorescence microscopy of yeast cells undergoing meiosis. This method allows tracking of individual cells over extended periods of time through every stage of the meiotic transformation while minimizing phototoxicity and sustaining conditions that support meiotic growth.

The conserved Cys-X1-X2-Cys motif present in the TtcA protein is required for the thiolation of cytidine in position 32 of tRNA from Salmonella enterica serovar Typhimurium.

The modified nucleoside 2-thiocytidine (s(2)C) has so far been found in tRNA from organisms belonging to the phylogenetic domains Archaea and Bacteria. In the bacteria Escherichia coli and Salmonella enterica serovar Typhimurium, s(2)C is present in position 32 of only four tRNA species-, and. An in-frame deletion of an S. enterica gene (designated ttcA, for "two-thio-cytidine") was constructed, and such a mutant has no detectable s(2)C in its tRNA. The TtcA protein family is characterized by the existence of both a PP-loop and a Cys-X(1)-X(2)-Cys motif in the central region of the protein but can be divided into two distinct groups based on the presence and location of additional Cys-X(1)-X(2)-Cys motifs in terminal regions of the sequence. Mutant analysis showed that both cysteines in this central conserved Cys-X(1)-X(2)-Cys motif are required for the formation of s(2)C. The DeltattcA1 mutant grows at the same rate as the congenic wild-type strain, and no growth disadvantage caused by the lack of s(2)C was observed in a mixed-population experiment. Lack of s(2)C32 did not reduce the selection rate at the ribosomal aminoacyl-tRNA site (A-site) for at any of its cognate CGN codons, whereas A-site selection at AGG by was dependent on the presence of s(2)C32. The presence of s(2)C32 in peptidyl- or in peptidyl- interfered with decoding in the A-site. The presence of s(2)C32 in decreased the rate of translation of the CGA codon but not that of the CGU codon.