Yeast calmodulin and a conserved nuclear protein participate in the in vivo binding of a matrix association region.

Chromatin becomes reorganized during mitosis each cell cycle. To identify genes potentially involved in these supramolecular events, we have used a colony-color assay to screen temperature-sensitive mutants of Saccharomyces cerevisiae. When a sequence that mediates attachment to the nuclear matrix in vitro was inserted into the GAL1 promoter of a lacZ fusion gene, beta-galactosidase synthesis was inhibited. This observation permitted screening for temperature-sensitive-inducible mutants on 5-bromo-4-chloro-3-indolyl beta-D-galactoside plates. Only 1 of 20 complementation groups of newly isolated mutants exhibited temperature-sensitive inducibility for the matrix association region but not for control CEN3 or STE6 inserts--a cmd1 mutant in which the last 7 amino acids of calmodulin were truncated by an ochre termination codon. Another mutant (smi1) exhibited a rare phenotype at the nonpermissive condition, which included S phase and budding arrest. We cloned and sequenced the SMI1 gene, which encodes a 57-kDa polypeptide with evolutionarily conserved epitope(s) found in mammalian cell nuclei. Thus, we provide evidence for involvement of calmodulin and another conserved protein in the in vivo binding of a matrix association region.

Basal-level expression of the yeast HSP82 gene requires a heat shock regulatory element.

Previous studies have shown that heat shock factor is constitutively bound to heat shock elements in Saccharomyces cerevisiae. We demonstrate that mutation of the heat shock element closest to the TATA box of the yeast HSP82 promoter abolishes basal-level transcription without markedly affecting inducibility. The mutated heat shock element no longer bound putative heat shock factor, either in vitro or in vivo, but still resided within a nuclease-hypersensitive site in the chromatin. Thus, constitutive binding of heat shock factor to heat shock elements in S. cerevisiae appears to functionally direct basal-level transcription.

Molecular cloning of a cDNA complementary to a UDP-glucose pyrophosphorylase mRNA of dictyostelium discoideum.

Uridine diphosphoglucose pyrophosphorylase (UTP: -alpha-D-glucose-1-phosphate uridyltransferase, EC 2.7.7.9) is an essential enzyme for normal development of Dictyostelium discoideum and its specific activity increases 3- to 10-fold by the later stages of development. Previous experiments have shown that additional forms of the enzyme appear concomitantly with this increase and that two uridine diphosphoglucose pyrophosphorylase (UDPGP) polypeptides are immunoprecipitated from the in vitro translation products of total cellular RNA at any stage of development (B. F. Fishel, R. E. Manrow and R. P. Dottin, 1982, Dev. Biol. 92, 175-187). Using an in vitro translation-immunoprecipitation assay of UDPGP mRNA, we show that an increase in the amount of translatable mRNA is correlated with the accumulation of enzyme during development. A cDNA bank was constructed from a mRNA population that had been enriched for UDPGP mRNA by size fractionation on sucrose gradients containing methylmercuric hydroxide (C. W. Schweinfest, R. W. Kwiatkowski, and R. P. Dottin, 1982, Proc. Natl. Acad. Sci. USA 79, 4997-5000). A 1.8-Kb cDNA complementary to a UDPGP mRNA was identified after screening the bank by hybridization selection and translation. Only the mRNA encoding the higher molecular weight in vitro translation product is hybrid selected by this cDNA. In hybrid-arrested translation experiments, the coding strand of this cDNA selectively inhibits the translation of only one of the two in vitro translation products. Therefore, there are two distinct UDPGP mRNAs.