Chun Ren Lim

The Saccharomyces cerevisiae RuvB-like Protein, Tih2p, Is Required for Cell Cycle Progression and RNA Polymerase II-directed Transcription

Two highly conserved RuvB-like putative DNA helicases, p47/TIP49b and p50/TIP49a, have been identified in the eukaryotes. Here, we study the function of Saccharomyces cerevisiae TIH2, which corresponds to mammalian p47/TIP49b. Tih2p is required for vegetative cell growth and localizes in the nucleus. Immunoprecipitation analysis revealed that Tih2p tightly interacts with Tih1p, the counterpart of mammalian p50/TIP49a, which has been shown to interact with the TATA-binding protein and the RNA polymerase II holoenzyme complex. Furthermore, the mutational study of the Walker A motif, which is required for nucleotide binding and hydrolysis, showed that this motif plays indispensable roles in the function of Tih2p. When a temperature-sensitive tih2 mutant,tih2–160, was incubated at the nonpermissive temperature, cells were rapidly arrested in the G1 phase. Northern blot analysis revealed that Tih2p is required for transcription of G1 cyclin and of several ribosomal protein genes. The similarities between the mutant phenotypes of tih2–160 and those of taf145 mutants suggest a role for TIH2in the regulation of RNA polymerase II-directed transcription.

Loss of Hsp70-Hsp40 Chaperone Activity Causes Abnormal Nuclear Distribution and Aberrant Microtubule Formation in M-phase of Saccharomyces cerevisiae

The 70-kDa heat shock proteins, hsp70, are highly conserved among both prokaryotes and eukaryotes, and function as chaperones in diverse cellular processes. To elucidate the function of the yeast cytosolic hsp70 Ssa1p in vivo, we characterized aSaccharomyces cerevisiae ssa1 temperature-sensitive mutant (ssa1-134). After shifting to the restrictive temperature (37 °C), ssa1-134 mutant cells showed abnormal distribution of nuclei and accumulated as large-budded cells with a 2n DNA content. We observed more prominent mutant phenotypes using nocodazole-synchronized cells: when cells were incubated at the restrictive temperature following nocodazole treatment, viability was rapidly lost and abnormal arrays of bent microtubules were formed. Chemical cross-linking and immunoprecipitation analyses revealed that the interaction of mutant Ssa1p with Ydj1p (cytosolic DnaJhomologue in yeast) was much weaker compared with wild-type Ssa1p. These results suggest that Ssa1p and Ydj1p chaperone activities play important roles in the regulation of microtubule formation in M phase. In support of this idea, a ydj1 null mutant at the restrictive temperature was found to exhibit more prominent phenotypes than ssa1-134. Furthermore, both ssa1-134 andydj1 null mutant cells exhibited greater sensitivity to anti-microtubule drugs. Finally, the observation that SSA1and YDJ1 interact genetically with a γ-tubulin,TUB4, supports the idea that they play a role in the regulation of microtubule formation.

Psychophysiological stress-regulated gene expression in mice

Eight genes showed significant changes in expression in mice under psychophysiological stress provided by cage‐restraint and water‐immersion. The transcription level of most of these genes was affected in all the tissues analyzed, and some of them were responsive genes in several different stress systems. Peculiarly, the expression level of one gene, cdc2‐like kinase 1 (CLK1), was reduced only in the brain, while the balance of partially‐ and alternatively‐spliced CLK1 mRNA species changed in all the tissues including the brain. These results suggest that some stress‐response mechanisms, including transcriptional and post‐transcriptional events, are coordinated in the whole body in mice under psychophysiological stress.

[31] S147P green fluorescent protein: A less thermosensitive green fluorescent protein variant

Publisher Summary The efficiency with which newly synthesized green fluorescent protein (GFP) polypeptides mature into the fluorescent active form depends largely on the temperature at which the cells or organisms expressing the gene are cultured. As the culture temperature increases, the maturation rate is retarded. This temperature sensitivity exhibited by the GFP molecule causes some difficulty in the practical use of GFP. This chapter discusses the basis of this problem and the way it may be addressed by use of a mutation, S147P, which alleviates the temperature sensitivity of the GFP molecule. The results here show that the S147P mutation alters some properties of GFP, including increased maturation efficiency at 37 ° and a 5-nm shift in the peak of excitation. Three-dimensional structural analysis of GFP shows that the Tyr-145 and His-148 amino acid residues near Ser-147 are in close proximity to the chromophore, and it is likely that the environment around the chromophore is significantly altered by the S147P mutation.