A Sampling of the Yeast Proteome

In this study, we examined yeast proteins by two-dimensional (2D) gel electrophoresis and gathered quantitative information from about 1,400 spots. We found that there is an enormous range of protein abundance and, for identified spots, a good correlation between protein abundance, mRNA abundance, and codon bias. For each molecule of well-translated mRNA, there were about 4,000 molecules of protein. The relative abundance of proteins was measured in glucose and ethanol media. Protein turnover was examined and found to be insignificant for abundant proteins. Some phosphoproteins were identified. The behavior of proteins in differential centrifugation experiments was examined. Such experiments with 2D gels can give a global view of the yeast proteome.     

Genetics,Morphology, Advertisement Calls,and Historical Records Distinguish Six New Polyploid Species of African Clawed Frog (Xenopus,Pipidae) from West and Central Africa

African clawed frogs, genus Xenopus, are extraordinary among vertebrates in the diversity of their polyploid species and the high number of independent polyploidization events that occurred during their diversification. Here we update current understanding of the evolutionary history of this group and describe six new species from west and central sub-Saharan Africa, including four tetraploids and two dodecaploids. We provide information on molecular variation, morphology, karyotypes, vocalizations, and estimated geographic ranges, which support the distinctiveness of these new species. We resurrect Xenopus calcaratus from synonymy of Xenopus tropicalis and refer populations from Bioko Island and coastal Cameroon (near Mt. Cameroon) to this species. To facilitate comparisons to the new species, we also provide comments on the type specimens, morphology, and distributions of X. epitropicalis, X. tropicalis, and X. fraseri. This includes significantly restricted application of the names X. fraseri and X. epitropicalis, the first of which we argue is known definitively only from type specimens and possibly one other specimen. Inferring the evolutionary histories of these new species allows refinement of species groups within Xenopus and leads to our recognition of two subgenera (Xenopus and Silurana) and three species groups within the subgenus Xenopus (amieti, laevis, and muelleri species groups).     

A single-molecule method for the quantitation of microRNA gene expression

MicroRNAs (miRNA) are short endogenous noncoding RNA molecules that regulate fundamental cellular processes such as cell differentiation, cell proliferation and apoptosis through modulation of gene expression. Critical to understanding the role of miRNAs in this regulation is a method to rapidly and accurately quantitate miRNA gene expression. Existing methods lack sensitivity, specificity and typically require upfront enrichment, ligation and/or amplification steps. The Direct miRNA assay hybridizes two spectrally distinguishable fluorescent locked nucleic acid (LNA)-DNA oligonucleotide probes to the miRNA of interest, and then tagged molecules are directly counted on a single-molecule detection instrument. In this study, we show the assay is sensitive to femtomolar concentrations of miRNA (500 fM), has a three-log linear dynamic range and is capable of distinguishing among miRNA family members. Using this technology, we quantified expression of 45 human miRNAs within 16 different tissues, yielding a quantitative differential expression profile that correlates and expands upon published results.     

A NASP (N1/N2)-related protein, Sim3, binds CENP-A and is required for its deposition at fission yeast centromeres

A defining feature of centromeres is the presence of the histone H3 variant CENP-A(Cnp1). It is not known how CENP-A(Cnp1) is specifically delivered to, and assembled into, centromeric chromatin. Through a screen for factors involved in kinetochore integrity in fission yeast, we identified Sim3. Sim3 is homologous to known histone binding proteins NASP(Human) and N1/N2(Xenopus) and aligns with Hif1(S. cerevisiae), defining the SHNi-TPR family. Sim3 is distributed throughout the nucleoplasm, yet it associates with CENP-A(Cnp1) and also binds H3. Cells defective in Sim3 function have reduced levels of CENP-A(Cnp1) at centromeres (and increased H3) and display chromosome segregation defects. Sim3 is required to allow newly synthesized CENP-A(Cnp1) to accumulate at centromeres in S and G2 phase-arrested cells in a replication-independent mechanism. We propose that one function of Sim3 is to act as an escort that hands off CENP-A(Cnp1) to chromatin assembly factors, allowing its incorporation into centromeric chromatin.     

Pheromone trap for the eastern tent caterpillar moth

The discovery that the eastern tent caterpillar Malacosoma americanum (F.) causes mare reproductive loss syndrome (MRLS), and thus has the potential to continue to result in major economic losses to the equine industry of Kentucky, has resulted in an intensive effort to identify practical means to monitor and control this defoliator, including these experiments to optimize a sex pheromone trap for this pest. A pheromone-baited delta trap with a large opening, such as InterceptST Delta, was more effective than other tested traps. Orange delta traps caught more moths than other tested colors. ETC males are caught at all tested heights within the tree canopy. For monitoring flights, setting traps at 1.5 m would allow easy counting of moths. A 9:1 blend of (E,Z)-5,7-dodecadienal (ETC-Ald) and (E,Z)-5,7-dodecadienol (ETC-OH) was most effective in capturing males. Increasing loading doses of a 3:1 blend (Ald:OH) resulted in the capture of increasing numbers of moths, but a 9:1 blend was more effective than 3:1 blend even at a nine-fold lower loading rate. Pheromone-impregnated white septa caught more moths than gray septa at the same loading dose. The advantages and limitations of using pheromone traps for monitoring M. americanum are discussed.     

Isolation of specific tRNAs using an ionic-hydrophobic mixed-mode chromatographic matrix

The coating of a C18-reversed-phase high performance liquid chromatography support (octadecylsilyl-Hypersil) with a tetraalkylammonium salt (methyltrioctylammonium chloride) produces a chromatographic matrix with both ionic and hydrophobic character. Using this material oligonucleotides and tRNAs can be separated with high resolution. The observed resolution is in part due to the apparent lack of diffusion processes occurring during chromatography with this matrix. Some tRNAs can be obtained in high purity from a bulk tRNA mixture after a single chromatographic step. In general it is more efficient to use the matrix as the last step of a purification procedure for a particular tRNA. A two-step procedure is described which allows, in some cases, the isolation of small quantities of specific tRNA isoacceptors.     

Effects of a Single Histoplasmin Skin Test on the Serological Diagnosis of Histoplasmosis

Numerous reports have indicated that a single histoplasmin skin test may stimulate humoral antibodies to Histoplasma capsulatum antigens in histoplasmin-hypersensitive individuals. Although these investigations concur that antibody elevations are evoked, they vary in the reported degree of incidence and response induced, and they cast doubt on the interpretation of serological tests in the diagnosis of histoplasmosis. Histoplasmin-hypersensitive subjects (114) were bled prior to administration of the skin test, 2 days later, at the time this test was read, and 15 and 30 days after testing. No significant antibody titers were observed at 2 days. At 15- and 30-day intervals, only 17 (15%) of the subjects demonstrated circulating antibodies. All 17 showed agar gel bands; 5 demonstrated no complement-fixation (CF) titers, 10 produced CF antibodies ranging from 1:8 to 1:16, and 2 demonstrated titers of 1:32. The data suggest that skin testing does not interfere significantly with antibody levels in sera drawn approximately 2 days after administration of antigen. However, since titers as high as 1:32 were obtained at later intervals, such reactions should be evaluated cautiously and only after consideration of clinical findings.     

Mitral stenosis with posterior diastolic movement of posterior leaflet.

The echocardiographic diagnosis of mitral stenosis depends in part on the demonstration of abnormal posterior leaflet movement to distinguish it from other conditions that similarly affect anterior leaflet motion. In mitral stenosis the posterior leaflet has been shown to move anteriorly in diastole rather than in the normal posterior direction. A patient presented with clinical evidence of moderate mitral stenosis. The anterior leaflet echo was typical but the posterior leaflet showed posterior diastolic movement. At catheterization moderate mitral stenosis was confirmed. To our knowledge this is the first report of the echocardiographic demonstration of posterior diastolic movement of the posterior mitral leaflet in documented mitral stenosis.