Genome-wide analysis of barcoded Saccharomyces cerevisiae gene-deletion mutants in pooled cultures

The availability of a near-complete (96%) collection of gene-deletion mutants in Saccharomyces cerevisiae greatly facilitates the systematic analyses of gene function in yeast. The unique 20 bp DNA 'barcodes' or 'tags' in each deletion strain enable the individual fitness of thousands of deletion mutants to be resolved from a single pooled culture. Here, we present protocols for the study of pooled cultures of tagged yeast deletion mutants with a tag microarray. This process involves five main steps: pooled growth, isolation of genomic DNA, PCR amplification of the barcodes, array hybridization and data analysis. Pooled deletion screening can be used to study gene function, uncover a compound's mode of action and identify drug targets. In addition to these applications, the general method of studying pooled samples with barcode arrays can also be adapted for use with other types of samples, such as mutant collections in other organisms, short interfering RNA vectors and molecular inversion probes.     

Rous sarcoma virus-transformed baby hamster kidney cells express higher levels of asparagine-linked tri- and tetraantennary glycopeptides containing [GlcNAc-beta (1,6)Man-alpha (1,6)Man] and poly-N-acetyllactosamine sequences than baby hamster kidney cells

The alterations in complex-type N-linked oligosaccharides that can occur when an animal cell line is transformed by two dissimilar viruses were examined by comparing the N-linked oligosaccharides of baby hamster kidney (BHK) cells, metabolically radiolabeled with [2-3H]mannose, to the same class of oligosaccharides from BHK cells separately transformed by Rous sarcoma virus (RS-BHK), an RNA retrovirus, and polyoma virus (PY-BHK), a DNA papovavirus. Based on experiments that utilized serial lectin affinity chromatography, glycosidase digestions, and methylation analyses, both RS-BHK and PY-BHK cells demonstrated a significant increase in the relative amounts of tri- and tetraantennary complex-type N-linked oligosaccharides containing the branching sequence, [GlcNAc-beta(1,6)Man-alpha(1,6)Man], compared to the nontransformed BHK cells. In addition, almost all of the poly-N-acetyllactosamine sequence, [GlcNAc-beta(1,3)-Gal-beta(1,4)], was expressed on the tri- and tetraantennary N-linked oligosaccharides from BHK and RS-BHK cells that contain the sequence, [GlcNAc-beta(1,6)Man-beta(1,6)Man]. The increase in the relative amounts of this latter sequence in the transformed cells, therefore, most likely results in an increase in the amount of poly-N-acetyllactosamine sequence on the N-linked glycopeptides of these cells. The analysis of the degree of sialylation of the complex-type N-linked oligosaccharides from BHK and RS-BHK cells by ion exchange chromatography revealed no apparent differences, and in both of these cell types approximately 3% of the glycopeptide fraction radiolabeled with mannose was recovered in a highly negatively charged fraction that was identified by keratanase digestion to be keratan sulfate.     

Inhibition of a specific N-glycosylation activity results in attenuation of breast carcinoma cell invasiveness-related phenotypes: inhibition of epidermal growth factor-induced dephosphorylation of focal adhesion kinase

Changes in the expression of glycosyltransferases that branch N-linked glycans can alter the function of several types of cell surface receptors and a glucose transporter. To study in detail the mechanisms by which aberrant N-glycosylation caused by altered N-acetylglucosaminyltransferase V(GnT-V, GnT-Va, and Mgat5a) expression can regulate the invasiveness-related phenotypes found in some carcinomas, we utilized specific small interfering RNA (siRNA) to selectively knock down GnT-V expression in the highly metastatic and invasive human breast carcinoma cell line, MDA-MB231. Knockdown of GnT-V by siRNA expression had no effect on epidermal growth factor receptor expression levels but lowered expression of N-linked beta(1,6)-branching on epidermal growth factor receptor, as expected. Compared with control cells, knockdown of GnT-V caused significant inhibition of the morphological changes and cell detachment from matrix that is normally seen after stimulation with epidermal growth factor (EGF). Decreased expression of GnT-V caused a marked inhibition of EGF-induced dephosphorylation of focal adhesion kinase (FAK), consistent with the lack of cell morphology changes in the cells expressing GnT-V siRNA. The attenuation of EGF-mediated phosphorylation and activation of the tyrosine phosphatase SHP-2 was dramatically observed in GnT-V knockdown cells, and these effects could be rescued by reintroduction of GnT-V into these cells, indicating that reduced EGF-mediated activation of SHP-2 was GnT-V related. Concomitantly, knockdown of GnT-V caused reduced EGF-mediated ERK signaling and tumor cell invasiveness-related phenotypes, including effects on actin rearrangement and cell motility. No changes in EGF binding were observed, however, after knockdown of GnT-V. Our results demonstrate that decreased GnT-V activity due to siRNA expression in human breast carcinoma cells resulted in an inhibition of EGF-stimulated SHP-2 activation and, consequently, caused attenuation of the dephosphorylation of FAK induced by EGF. These effects suppressed EGF-mediated downstream signaling and invasiveness-related phenotypes and suggest GnT-V as a potential therapeutic target.     

Effects of mitogen-activated protein kinases on nuclear protein import

ERK-2 MAP kinase activation induces inhibitory effects on nuclear protein import in vascular smooth muscle cells. The mechanism and characteristics of this effect of ERK-2 were investigated. An unusual dose-dependent effect of ERK-2 on nuclear protein import was identified. At higher concentrations (1 microg/mL) of ERK-2, nuclear protein import was stimulated, whereas lower concentrations (0.04 microg/mL) inhibited import. Intermediate concentrations exerted intermediate effects. The stimulatory and inhibitory effects at the 2 different ERK-2 concentrations were observed in both conventional, permeabilized cell assays of nuclear protein import and with in situ microinjection of smooth muscle cells. The biphasic effects of ERK-2 on import were also found for the other 2 members of the MAPK family, p38 and JNK. RanGAP was identified by structural analysis as a candidate target protein responsible for mediating the effects of ERK-2. After pretreatment with high concentrations of ERK-2, RanGAP activity was significantly increased by approximately 50%. In contrast, low concentrations of ERK-2 significantly attenuated RanGAP activity. These results demonstrate that all 3 members of the MAPK family can alter nuclear protein import in opposite directions depending upon the concentration of ERK-2 used. RanGAP represents the MAP kinase target whereby nuclear transport can be stimulated or inhibited.     

Allowing for random errors in radiation dose estimates for the atomic bomb survivor data

The presence of random errors in the individual radiation dose estimates for the A-bomb survivors causes underestimation of radiation effects in dose-response analyses, and also distorts the shape of dose-response curves. Statistical methods are presented which will adjust for these biases, provided that a valid statistical model for the dose estimation errors is used. Emphasis is on clarifying some rather subtle statistical issues. For most of this development the distinction between radiation dose and exposure is not critical. The proposed methods involve downward adjustment of dose estimates, but this does not imply that the dosimetry system is faulty. Rather, this is a part of the dose-response analysis required to remove biases in the risk estimates. The primary focus of this report is on linear dose-response models, but methods for linear-quadratic models are also considered briefly. Some plausible models for the dose estimation errors are considered, which have typical errors in a range of 30-40% of the true values, and sensitivity analysis of the resulting bias corrections is provided. It is found that for these error models the resulting estimates of excess cancer risk based on linear models are about 6-17% greater than estimates that make no allowance for dose estimation errors. This increase in risk estimates is reduced to about 4-11% if, as has often been done recently, survivors with dose estimates above 4 Gy are eliminated from the analysis.