The conjugation of three amine reactive fluorescent probes, each containing the fluorophore fluorescein but different reactive moieties, was compared using the protein myoglobin and the amino acid L-lysine as reagents. The three different reactive moieties were an isothiocyanate group (FITC), a succinimidyl ester group (CFSE), and a dichlorotriazine group (DTAF). The relative performance was based on the degree of conjugation to myoglobin, the rate of reaction, freedom from hydrolysis, and the stability of a conjugate with lysine. Performance was evaluated by separating the conjugation reaction reagents and products on-line, using capillary zone electrophoresis, and assessing relative amounts by absorbance detection. Each of the reactive probes demonstrated the ability to achieve a similar degree of conjugation, which depended mostly on allowed conjugated reaction time, and a rate of conjugation that rendered hydrolysis of the reactive moiety insignificant. For the relative rate of conjugation between probes and stability of the resulting conjugate, CFSE demonstrated superior performance, followed by DTAF and then FITC, for both the protein myoglobin and the amino acid L-lysine. The FITC conjugation reaction was much easier to control, however, which may be significant for applications that require a precise degree of conjugation. With regard to conjugate-bond stability, the FITC conjugate demonstrated inferior performance when subjected to incubation at 37 degrees C.

Saccharomyces cerevisiae Scc2 binds Scc4 to form an essential complex that loads cohesin onto chromosomes. The prevalence of Scc2 orthologs in eukaryotes emphasizes a conserved role in regulating sister chromatid cohesion, but homologs of Scc4 have not hitherto been identified outside certain fungi. Some metazoan orthologs of Scc2 were initially identified as developmental gene regulators, such as Drosophila Nipped-B, a regulator of cut and Ultrabithorax, and delangin, a protein mutant in Cornelia de Lange syndrome. We show that delangin and Nipped-B bind previously unstudied human and fly orthologs of Caenorhabditis elegans MAU-2, a non-axis-specific guidance factor for migrating cells and axons. PSI-BLAST shows that Scc4 is evolutionarily related to metazoan MAU-2 sequences, with the greatest homology evident in a short N-terminal domain, and protein–protein interaction studies map the site of interaction between delangin and human MAU-2 to the N-terminal regions of both proteins. Short interfering RNA knockdown of human MAU-2 in HeLa cells resulted in precocious sister chromatid separation and in impaired loading of cohesin onto chromatin, indicating that it is functionally related to Scc4, and RNAi analyses show that MAU-2 regulates chromosome segregation in C. elegans embryos. Using antisense morpholino oligonucleotides to knock down Xenopus tropicalis delangin or MAU-2 in early embryos produced similar patterns of retarded growth and developmental defects. Our data show that sister chromatid cohesion in metazoans involves the formation of a complex similar to the Scc2-Scc4 interaction in the budding yeast. The very high degree of sequence conservation between Scc4 homologs in complex metazoans is consistent with increased selection pressure to conserve additional essential functions, such as regulation of cell and axon migration during development.

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Nutrient fluctuations in the cellular environment promote changes in cell metabolism and growth to adapt cell proliferation accordingly. The target of rapamycin (TOR) signalling network plays a key role in the coordination of growth and cell proliferation with the nutrient environment and, importantly, nutrient limitation reduces TOR complex 1 (TORC1) signalling. We have performed global quantitative fitness profiling of the collection of Schizosaccharomyces pombe strains from which non-essential genes have been deleted. We identified genes that regulate fitness when cells are grown in a nutrient-rich environment compared with minimal environments, with varying nitrogen sources including ammonium, glutamate and proline. In addition, we have performed the first global screen for genes that regulate fitness when both TORC1 and TORC2 signalling is reduced by Torin1. Analysis of genes whose deletions altered fitness when nutrients were limited, or when TOR signalling was compromised, identified a large number of genes that regulate transmembrane transport, transcription and chromatin organization/regulation and vesicle-mediated transport. The ability to tolerate reduced TOR signalling placed demands upon a large number of biological processes including autophagy, mRNA metabolic processing and nucleocytoplasmic transport. Importantly, novel biological processes and all processes known to be regulated by TOR were identified in our screens. In addition, deletion of 62 genes conserved in humans gave rise to strong sensitivity or resistance to Torin1, and 29 of these 62 genes have novel links to TOR signalling. The identification of chromatin and transcriptional regulation, nutritional uptake and transport pathways in this powerful genetic model now paves the way for a molecular understanding of how cells adapt to the chronic and acute fluctuations in nutrient supply that all eukaryotes experience at some stage, and which is a key feature of cancer cells within solid tumours.