In this paper we show that a 211-base pair segment of CEN3 DNA is sufficient to confer wild-type centromere function in the yeast Saccharomyces cerevisiae. We used site-directed mutagenesis of the 211-base pair fragment to examine the sequence-specific functional requirements of a conserved 11-base pair segment of centromere DNA, element III (5'-TGTTTACCGAA-3'). Element III is the most highly conserved of the centromeric DNA sequences, differing by only a single adenine * thymine base pair among the four centromere DNAs sequenced thus far. All of the element III sequences contain specific cytosine-guanine base pairs, including a 5'-CCG-3' arrangement, which we targeted for single cytosine-to-thymine mutations by using sodium bisulfite. The effects of element III mutations on plasmid and chromosome segregation were determined by mitotic stability assays. Conversion of CCG to CTG completely abolished centromere function both in plasmids and in chromosome III, whereas conversion of CCG to TCG decreased plasmid and chromosome stability moderately. The other two guanine * cytosine base pairs in element III could be independently converted to adenine * thymine base pairs without affecting plasmid or chromosome stability. We concluded that while some specific nucleotides within the conserved element III sequence are essential for proper centromere function, other conserved nucleotides can be changed.Saccharomyces cerevisiae is the only organism from which centromere DNA has been identified and sequenced. The centromere is a specialized segment of each chromosome which functions at each cell division to ensure proper chromosome segregation. Centromere DNA has been isolated and mapped to six different yeast chromosomes, CEN3 (8), CENII (12), CEN6 (27), CEN4 (33), CENS (21), and CEN14 (M. Neitz and J. Carbon, unpublished data). Four of these CEN DNAs have been sequenced and have been shown to contain three common features of sequence organization (5, 12, 27). All contain an extremely adeninie thymirne-rich region which is 82 to 89 base pairs (bp) long (element II) and is flanked on either side by short regions of partial homology, elements I and III. As Fig. 1 shows, the element III sequence is identical in CEN3 and CENIl and differs by only one adenine * thymine base pair in CEN4 and CEN6.At least three lines of evidence suggest that these sequence elements are involved in centromere function. Centromere plasmids containing the element I through III region are stably maintained in yeasts, and complete deletion of these sequences results in dramatic plasmid instability (3, 13). Plasmids containing two CEN DNAs (dicentrics) are unstable in mitosis and suffer deletions involving the conserved CEN sequence elements, suggesting that there is a physical interaction between the conserved CEN DNA and the spindle apparatus (23). Recently, Clarke and Carbon (9) demonstrated that an acentric chromosome which was generated by complete deletion of sequence elements I, II, and III from chromosome III was extremely unstable. Therefore,...

Yeast cell surface display is a powerful tool for expression and immobilization of biocatalytically active proteins on a unicellular eukaryote. Here bacterial carboxylesterase EstA from Burkholderia gladioli was covalently anchored into the cell wall of Saccharomyces cerevisiae by in-frame fusion to the endogenous yeast proteins Kre1p, Cwp2p, and Flo1p. When p-nitrophenyl acetate was used as a substrate, the esterase specific activities of yeast expressing the protein fusions were 103 mU mg−1 protein for Kre1/EstA/Cwp2p and 72 mU mg−1 protein for Kre1/EstA/Flo1p. In vivo cell wall targeting was confirmed by esterase solubilization after laminarinase treatment and immunofluorescence microscopy. EstA expression resulted in cell wall-associated esterase activities of 2.72 U mg−1 protein for Kre1/EstA/Cwp2p and 1.27 U mg−1 protein for Kre1/EstA/Flo1p. Furthermore, esterase display on the yeast cell surface enabled the cells to effectively grow on the esterase-dependent carbon source glycerol triacetate (Triacetin). In the case of Kre1/EstA/Flo1p, in vivo maturation within the yeast secretory pathway and final incorporation into the wall were further enhanced when there was constitutive activation of the unfolded protein response pathway. Our results demonstrate that esterase cell surface display in yeast, which, as shown here, is remarkably more effective than EstA surface display in Escherichia coli, can be further optimized by activating the protein folding machinery in the eukaryotic secretion pathway.

In this paper we show that a 211-base pair segment of CEN3 DNA is sufficient to confer wild-type centromere function in the yeast Saccharomyces cerevisiae. We used site-directed mutagenesis of the 211-base pair fragment to examine the sequence-specific functional requirements of a conserved 11-base pair segment of centromere DNA, element III (5'-TGATTTATCCGAA-3'). Element III is the most highly conserved of the centromeric DNA sequences, differing by only a single adenine X thymine base pair among the four centromere DNAs sequenced thus far. All of the element III sequences contain specific cytosine X guanine base pairs, including a 5'-CCG-3' arrangement, which we targeted for single cytosine-to-thymine mutations by using sodium bisulfite. The effects of element III mutations on plasmid and chromosome segregation were determined by mitotic stability assays. Conversion of CCG to CTG completely abolished centromere function both in plasmids and in chromosome III, whereas conversion of CCG to TCG decreased plasmid and chromosome stability moderately. The other two guanine X cytosine base pairs in element III could be independently converted to adenine X thymine base pairs without affecting plasmid or chromosome stability. We concluded that while some specific nucleotides within the conserved element III sequence are essential for proper centromere function, other conserved nucleotides can be changed.

Saccharomyces cerevisiae chromosome I has provided a vivid example of the "gene-number paradox." Although molecular studies have suggested that there are greater than 100 transcribed regions on the chromosome, classical genetic studies have identified only about 15 genes, including just 6 identified in intensive studies using Ts- lethal mutations. To help elucidate the reasons for this disparity, we have undertaken a detailed molecular analysis of a 34-kb segment of the left arm of the chromosome. This segment contains the four known genes CDC24, WHI1, CYC3 and PYK1 plus at least seven transcribed regions of unknown function. The 11 identified transcripts have a total length of approximately 25.9 kb, suggesting that greater than or equal to 75% of the DNA in this region is transcribed. Of the transcribed regions of unknown function, three are essential for viability on rich medium and three appear to be nonessential, as judged by the lethality or nonlethality of deletions constructed using integrative transformation methods. No obvious phenotypes were associated with the deletions in the apparently nonessential genes. However, two of these genes may have homologs elsewhere in the genome, as judged from the appearance of additional bands when DNA-DNA blot hybridizations were performed at reduced stringency. Taken together, the results provide further evidence that the limitations of classical genetic studies of chromosome I cannot be explained solely by a lack of genes, or even a lack of essential genes, on the chromosome.

The chelate Claisen rearrangement is a versatile tool for the stereoselective synthesis of β‐substituted γ,δ‐unsaturated amino acids, which can be converted into β‐substitutedaspartates by oxidative cleavage. These are ideal precursors for the synthesis of hydroxamate‐type MMP inhibitors.