Synthesis and biological activity of the four stereoisomers of 4-methyl-3-heptanol: main component of the aggregation pheromone of Scolytus amygdali.

Stereoisomers of 4-methyl-3-heptanol are major components of aggregation pheromones of bark beetles and trail pheromones of ants. Recently, (3S,4S)-4-methyl-3-heptanol (I) has been tentatively identified as the main component of the aggregation pheromone of the almond bark beetle, Scolytus amygdali (Coleoptera: Scolytidae). The four stereoisomers of 4-methyl-3-heptanol were prepared and bioassayed. Key steps included preparation of chiral 4-methyl-3-heptanones using SAMP and RAMP reagents, reduction to the corresponding alcohols, and stereospecific transesterification with vinyl acetate with lipase AK catalysis. In field tests, only (3S,4S)-4-methyl-3-heptanol attracted beetles in combination with the synergist (3S,4S)-4-methyl-3-hexanol, whereas (3R,4S)- and (3R,4R)-4-methyl-3-heptanols were inhibitory.

Fruiting body formation by Bacillus subtilis.

Spore formation by the bacterium Bacillus subtilis has long been studied as a model for cellular differentiation, but predominantly as a single cell. When analyzed within the context of highly structured, surface-associated communities (biofilms), spore formation was discovered to have heretofore unsuspected spatial organization. Initially, motile cells differentiated into aligned chains of attached cells that eventually produced aerial structures, or fruiting bodies, that served as preferential sites for sporulation. Fruiting body formation depended on regulatory genes required early in sporulation and on genes evidently needed for exopolysaccharide and surfactin production. The formation of aerial structures was robust in natural isolates but not in laboratory strains, an indication that multicellularity has been lost during domestication of B. subtilis. Other microbial differentiation processes long thought to involve only single cells could display the spatial organization characteristic of multicellular organisms when studied with recent natural isolates.

Genetic and physical interactions between factors involved in both cell cycle progression and pre-mRNA splicing in Saccharomyces cerevisiae.

The PRP17/CDC40 gene of Saccharomyces cerevisiae functions in two different cellular processes: pre-mRNA splicing and cell cycle progression. The Prp17/Cdc40 protein participates in the second step of the splicing reaction and, in addition, prp17/cdc40 mutant cells held at the restrictive temperature arrest in the G2 phase of the cell cycle. Here we describe the identification of nine genes that, when mutated, show synthetic lethality with the prp17/cdc40Delta allele. Six of these encode known splicing factors: Prp8p, Slu7p, Prp16p, Prp22p, Slt11p, and U2 snRNA. The other three, SYF1, SYF2, and SYF3, represent genes also involved in cell cycle progression and in pre-mRNA splicing. Syf1p and Syf3p are highly conserved proteins containing several copies of a repeated motif, which we term RTPR. This newly defined motif is shared by proteins involved in RNA processing and represents a subfamily of the known TPR (tetratricopeptide repeat) motif. Using two-hybrid interaction screens and biochemical analysis, we show that the SYF gene products interact with each other and with four other proteins: Isy1p, Cef1p, Prp22p, and Ntc20p. We discuss the role played by these proteins in splicing and cell cycle progression.

Functional analyses of interacting factors involved in both pre-mRNA splicing and cell cycle progression in Saccharomyces cerevisiae.

Through a genetic screen to search for factors that interact with Prp17/Cdc40p, a protein involved in both cell cycle progression and pre-mRNA splicing, we identify three novel factors, which we call Syf1p, Syf2p, and Syf3 (SYnthetic lethal with cdc Forty). Here we present evidence that all three proteins are spliceosome associated, that they associate weakly or transiently with U6 and U5 snRNAs, and that Syf1p and Syf3p (also known as Clf1p) are required for pre-mRNA splicing. In addition we show that depletion of Syf1p or Syf3p results in cell cycle arrest at the G2/M transition. Thus, like Prp17/Cdc40p, Syf1p and Syf3p are involved in two distinct cellular processes. We discuss the likelihood that Syf1p, Syf2p, and Syf3p are components of a protein complex that assembles into spliceosomes and also regulates cell cycle progression.

Extensive genetic interactions between PRP8 and PRP17/CDC40, two yeast genes involved in pre-mRNA splicing and cell cycle progression.

Biochemical and genetic experiments have shown that the PRP17 gene of the yeast Saccharomyces cerevisiae encodes a protein that plays a role during the second catalytic step of the splicing reaction. It was found recently that PRP17 is identical to the cell division cycle CDC40 gene. cdc40 mutants arrest at the restrictive temperature after the completion of DNA replication. Although the PRP17/CDC40 gene product is essential only at elevated temperatures, splicing intermediates accumulate in prp17 mutants even at the permissive temperature. In this report we describe extensive genetic interactions between PRP17/CDC40 and the PRP8 gene. PRP8 encodes a highly conserved U5 snRNP protein required for spliceosome assembly and for both catalytic steps of the splicing reaction. We show that mutations in the PRP8 gene are able to suppress the temperature-sensitive growth phenotype and the splicing defect conferred by the absence of the Prp17 protein. In addition, these mutations are capable of suppressing certain alterations in the conserved PyAG trinucleotide at the 3' splice junction, as detected by an ACT1-CUP1 splicing reporter system. Moreover, other PRP8 alleles exhibit synthetic lethality with the absence of Prp17p and show a reduced ability to splice an intron bearing an altered 3' splice junction. On the basis of these findings, we propose a model for the mode of interaction between the Prp8 and Prp17 proteins during the second catalytic step of the splicing reaction.

Efficient initiation of S-phase in yeast requires Cdc40p, a protein involved in pre-mRNA splicing.

The S. cerevisiae CDC40 gene was originally identified as a cell-division-specific gene that is essential only at elevated temperatures. Cells carrying mutations in this gene arrest with a large bud and a single nucleus with duplicated DNA content. Cdc40p is also required for spindle establishment or maintenance. Sequence analysis reveals that CDC40 is identical to PRP17, a gene involved in pre-mRNA splicing. In this paper, we show that Cdc40p is required at all temperatures for efficient entry into S-phase and that cell cycle arrest associated with cdc40 mutations is independent of all the known checkpoint mechanisms. Using immunofluorescence, we show that Cdc40p is localized to the nuclear membrane, weakly associated with the nuclear pore. Our results point to a link between cell cycle progression, pre-mRNA splicing, and mRNA export.

Identification and functional analysis of hPRP17, the human homologue of the PRP17/CDC40 yeast gene involved in splicing and cell cycle control.

The PRP17 gene of the yeast Saccharomyces cerevisiae encodes a protein that participates in the second step of the splicing reaction. It was found recently that the yeast PRP17 gene is identical to the cell division cycle CDC40 gene. The PRP17/CDC40 gene codes for a protein with several copies of the WD repeat, a motif found in a large family of proteins that play important roles in signal transduction, cell cycle progression, splicing, transcription, and development. In this report, we describe the identification of human, nematode, and fission yeast homologues of the PRP17/CDC40 gene of S. cerevisiae. The newly identified proteins share homology with the budding yeast protein throughout their entire sequence, with the similarity being greatest in the C-terminal two thirds that includes the conserved WD repeats. We show that a yeast-human chimera, carrying the C-terminal two thirds of the hPRP17 protein, is able to complement the cell cycle and splicing defects of a yeast prp17 mutant. Moreover, the yeast and yeast-human chimeric proteins co-precipitate the intron-exon 2 lariat intermediate and the intron lariat product, providing evidence that these proteins are spliceosome-associated. These results show the functional conservation of the Prp17 proteins in evolution and suggest that the second step of splicing takes place by a similar mechanism throughout eukaryotes.

The role of Saccharomyces cerevisiae Cdc40p in DNA replication and mitotic spindle formation and/or maintenance.

Successful progression through the cell cycle requires the coupling of mitotic spindle formation to DNA replication. In this report we present evidence suggesting that, in Saccharomyces cerevisiae, the CDC40 gene product is required to regulate both DNA replication and mitotic spindle formation. The deduced amino acid sequence of CDC40 (455 amino acids) contains four copies of a beta-transducin-like repeat. Cdc40p is essential only at elevated temperatures, as a complete deletion or a truncated protein (deletion of the C-terminal 217 amino acids in the cdc40-1 allele) results in normal vegetative growth at 23 degrees C, and cell cycle arrest at 36 degrees C. In the mitotic cell cycle Cdc40p is apparently required for at least two steps: (1) for entry into S phase (neither DNA synthesis, nor mitotic spindle formation occurs at 36 degrees C and (2) for completion of S-phase (cdc40::LEU2 cells cannot complete the cell cycle when returned to the permissive temperature in the presence of hydroxyurea). The role of Cdc40p as a regulatory protein linking DNA synthesis, spindle assembly/maintenance, and maturation promoting factor (MPF) activity is discussed.