Branched chain amino acid regulation of the ILV2 locus in Saccharomyces cerevisiae

Mutant regulatory loci of the branched pathway for the biosynthesis of isoleucine-valine and leucine were identified with the unusual phenotype of an amino acid dependent auxotrophy. Two mutant loci, bcs1 and bcs2, conferred branched chain amino acid sensitivity and showed independent segregation. Linkage studies defined bcs1 as a cis-acting regulatory site of ILV2 (SMR1). ILV2 upstream deletion analyses and high-copy transformation of the positive regulatory locus LEU3 ruled out the possibility of LEU3 protein binding palindromes mediating the branched chain amino acid dependent auxotrophy. In the presence of leucine and valine, the general amino acid control system (GCN4) was epistatic to bcs1 and bcs2, and under nonstarvation conditions GCN4 strains showed an increased acetolactate synthase activity over gcn4 strains. Thus in addition to general regulation of ILV2, GCN4 functions in basal level expression when the locus is subject to specific repression by pathway end product.     

Evidence forCis- andTrans-acting element coevolution of the 2-μm circle genome inSaccharomyces cerevisiae

Summary We compared the DNA sequence of the yeas 2-μm plasmidcis-actingSTB andtrans-actingREP1 partition loci of laboratory haploid and industrial amphiploid strains. Several industrial strains had a uniqueSTB sequence (type 1) sharing only 70% homology with laboratorySTB (type 2). Type 1 plasmids had a REP1 protein with 6–10% amino acid substitutions when compared to REP1 of type 2 plasmids. All 2-μm variants that shared a similarSTB consensus sequence exhibited a high degree ofREP1 nucleotide and amino acid sequence conservation. These observations suggest molecular coevolution oftrans-acting elements with cognate target DNA structure. Based on DNA sequencing and Southern hybridization analyses, we classified 2-μm variants into two main evolutionary lineages that differ atSTB as well asREP1 loci. The role of molecular coevolution in yeast intra- and interspecies plasmid evolution was discussed.     

Host plant preference based on salicylate chemistry in a willow leaf beetle (Chrysomela aeneicollis)

Summary Chrysomela aeneicollis (Coleoptera: Chrysomelidae) uses salicin from its host plant (Salix spp.) to produce a defensive secretion, salicylaldehyde. Because it requires salicin for this secretion, I predicted that C. aeneicollis should be attracted to willows which possess salicin and other salicylates. To test this prediction, I determined the host-plant preferences of C. aeneicollis among four potential hosts which occur in the Sierra Nevada range of eastern California. These species have very different salicylate chemistries but do not differ in nutritional quality for C. aeneicollis. In oviposition-preference tests, gravid females showed no preference between a salicylate-poor species, S. lutea, and a salicylate-rich species, S. orestera. However in feeding-choice tests, both larvae and adults preferred S. orestera over S. lutea. This preference was not affected by the species on which the larvae were reared. In other feeding tests, adults preferred S. orestera over two medium-salicylate species, S. boothi and S. geyeriana, regardless of which host species they had been feeding on in nature. In a final feeding test, adults were stimulated to feed by salicin itself. In nature, the relative abundances of C. aeneicollis adults and egg clutches among these species correspond to the adult feeding preference in the laboratory. Additionally, multiple regression analyses showed that adult abundance was not related to among-clone differences in leaf toughness or nutritional quality, but rather to salicin content and plant size. Thus for C. aeneicollis, both laboratory and field results demonstrate a preference for salicylate-rich willows which is partly responsible for the increased level of attack on them.     

Reduced plasma membrane permeability in a multiple cross-resistant strain of Saccharomyces cerevisiae.

Single nuclear gene inheritance was shown to be responsible for increased resistance to: eight diverse inhibitors of mitochondrial function (antimycin, carbonylcyanide-m-chlorophenylhydrazone, chloramphenicol, oligomycin, tetracycline, triethyltin bromide, triphenylmethylphosphonium bromide and triton-X-165); and an inhibitor of cytoplasmic protein synthesis (cycloheximide). Continuous monitoring of oxygen uptake during respiratory adaptation showed that anerobic pretreatment of resistant cells sensitized respiratory adaptation to chloramphenicol and antimycin. However, since a depression of mitochondrial function by catabolite repression did not result in sensitization to antimycin, alteration of the mitochondrial membrane does not appear to be responsible for resistance to mitochondrial inhibition. Alteration of cellular binding sites was not responsible for resistance since in vitro mitochondrial protein synthesis was sensitive to chloramphenicol and in vitro mitochondrial respiration was sensitive to oligomycin, carbonylcyanide-m-chlorophenylhydrazone, and antimycin. Autoradiography of an ethylacetate-ethanol extract of [14C]chloramphenicol-treated resistant cells indicated that resistance was not due to enzymatic modification of inhibitors. The maintenance of an antimycin-resistant respiration by protoplasts of resistant cells ruled out the involvement of the cell wall in cellular resistance. The reduced transport of [14C]chloramphenicol by resistant cells (1% of normal cells) indicated that a single nuclear gene mutation can alter the permeability of the plasma membrane to many diverse inhibitors.     

Comparative analysis of tandem repeats from hundreds of species reveals unique insights into centromere evolution

Background

Centromeres are essential for chromosome segregation, yet their DNA sequences evolve rapidly. In most animals and plants that have been studied, centromeres contain megabase-scale arrays of tandem repeats. Despite their importance, very little is known about the degree to which centromere tandem repeats share common properties between different species across different phyla. We used bioinformatic methods to identify high-copy tandem repeats from 282 species using publicly available genomic sequence and our own data.

Results

Our methods are compatible with all current sequencing technologies. Long Pacific Biosciences sequence reads allowed us to find tandem repeat monomers up to 1,419 bp. We assumed that the most abundant tandem repeat is the centromere DNA, which was true for most species whose centromeres have been previously characterized, suggesting this is a general property of genomes. High-copy centromere tandem repeats were found in almost all animal and plant genomes, but repeat monomers were highly variable in sequence composition and length. Furthermore, phylogenetic analysis of sequence homology showed little evidence of sequence conservation beyond approximately 50 million years of divergence. We find that despite an overall lack of sequence conservation, centromere tandem repeats from diverse species showed similar modes of evolution.

Conclusions

While centromere position in most eukaryotes is epigenetically determined, our results indicate that tandem repeats are highly prevalent at centromeres of both animal and plant genomes. This suggests a functional role for such repeats, perhaps in promoting concerted evolution of centromere DNA across chromosomes.     

Proteomic characterization of the interstitial fluid perfusing the breast tumor microenvironment: a novel resource for biomarker and therapeutic target discovery

Clinical cancer proteomics aims at the identification of markers for early detection and predictive purposes, as well as to provide novel targets for drug discovery and therapeutic intervention. Proteomics-based analysis of traditional sources of biomarkers, such as serum, plasma, or tissue lyzates, has resulted in a wealth of information and the finding of several potential tumor biomarkers. However, many of these markers have shown limited usefulness in a clinical setting, underscoring the need for new clinically relevant sources. Here we present a novel and highly promising source of biomarkers, the tumor interstitial fluid (TIF) that perfuses the breast tumor microenvironment. We collected TIFs from small pieces of freshly dissected invasive breast carcinomas and analyzed them by two-dimensional polyacrylamide gel electrophoresis in combination with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, Western immunoblotting, as well as by cytokine-specific antibody arrays. This approach provided for the first time a snapshot of the protein components of the TIF, which we show consists of more than one thousand proteins--either secreted, shed by membrane vesicles, or externalized due to cell death--produced by the complex network of cell types that make up the tumor microenvironment. So far, we have identified 267 primary translation products including, but not limited to, proteins involved in cell proliferation, invasion, angiogenesis, metastasis, inflammation, protein synthesis, energy metabolism, oxidative stress, the actin cytoskeleton assembly, protein folding, and transport. As expected, the TIF contained several classical serum proteins. Considering that the protein composition of the TIF reflects the physiological and pathological state of the tissue, it should provide a new and potentially rich resource for diagnostic biomarker discovery and for identifying more selective targets for therapeutic intervention.     

W206R]-procaspase 3: an inactivatable substrate for caspase 8.

We report here the cloning and high-level expression of a soluble proform of human caspase 3 (Ser(24)-H(277)) engineered to contain a short stretch of N-terminal sequence (MTISDSPREQD) from the prosegment of procaspase 8 and a C-terminal heptahistidine tag. The precursor protein isolated from extracts of recombinant Escherichia coli by immobilized metal-ion affinity chromatography was predominantly unprocessed and migrated as a 32-kDa polypeptide on sodium dodecyl sulfate-polyacrylamide gels. Incubation of this protein with recombinant human caspase 8 produced fragments characteristic of the properly processed caspase 3, but the product was inactive. Amino-terminal sequence analysis of the caspase 3 polypeptides proved that caspase 8 had specifically cleaved the Asp(175)-Ser(176) bond to yield the expected p18 and p12 subunits, with partial cleavage at the Asp(28)-Ser(29) bond to release the prosegment. The lack of caspase 3 activity was found to be the result of a fortuitous mutation in which Trp(206) in the S4 subsite was replaced by arginine (W206R). This mutant procaspase 3, which we call m-pro3, serves as a useful reagent with which to test the efficacy of caspase 8 inhibitors in blocking processing of the natural polypeptide substrate of this enzyme and may be valuable as a source of "proenzyme" for crystallographic analysis.     

Allele frequency shifts in response to climate change and physiological consequences of allozyme variation in a montane insect

Rapid changes in climate may impose strong selective pressures on organisms. Evolutionary responses to climate change have been observed in natural populations, yet no example has been documented for a metabolic enzyme locus. Furthermore, few studies have linked physiological responses to stress with allozyme genotypic variation. We quantified changes in allele frequency between 1988 and 1996 at three allozyme loci (isocitrate dehydrogenase, Idh; phosphoglucose isomerase, Pgi; and phosphoglucomutase, Pgm) for the leaf beetle Chrysomela aeneicollis in the Bishop Creek region of the Sierra Nevada of California (2900-3300 m). Beetles often experience high daytime (> 32 degrees C) and extremely low nighttime (< -5 degrees C) temperatures during summer. Bishop Creek weather station data indicated that conditions were unusually dry before 1988, and that conditions were cool and wet during the years preceding the 1996 collection. We found directional changes in allele frequency at Pgi (11% increase in the Pgi-1 allele), but not at Idh or Pgm. We also found that physiological response to thermal extremes depended on Pgi genotype. Pgi 1-1 individuals induced expression of a 70-kD heat shock protein (HSP) at lower temperatures than 1-4 or 4-4 individuals, and 1-1 individuals expressed higher levels of HSP70 after laboratory exposure to temperatures routinely experienced in nature. Survival after nighttime laboratory exposure to subzero temperatures depended on gender, previous exposure to cold, and Pgi genotype. Females expressed higher levels of HSP70 than males after exposure to heat, and recovery by female Pgi 1-1 homozygotes after exposure to cold (-5 degrees C) was significantly better than 1-4 or 4-4 genotypes. These data suggest that the cooler climate of the mid-1990s may have caused an increase in frequency of the Pgi-1 allele, due to a more robust physiological response to cold by Pgi 1-1 and 1-4 genotypes.     

Direct interaction of soluble human recombinant tau protein with Abeta 1-42 results in tau aggregation and hyperphosphorylation by tau protein kinase II

We report here that aggregated beta-amyloid (Abeta) 1-42 promotes tau aggregation in vitro in a dose-dependent manner. When Abeta-mediated aggregated tau was used as a substrate for tau protein kinase II (TPK II), an 8-fold increase in the rate of TPK II-mediated tau phosphorylation was observed. The extent of TPK II-dependent tau phosphorylation increased as a function of time and Abeta 1-42 concentration, and hyperphosphorylated tau was found to be decorated with an Alzheimer's disease-related phosphoepitope (P-Thr-231). In HEK 293 cells co-expressing CT-100 amyloid precursor protein and tau, the release of Abeta 1-42 from these cells was impaired. Taken together, these in vitro results suggest that Abeta 1-42 promotes both tau aggregation and hyperphosphorylation.