News & Highlights: A recent collabaration between YRC researcher John Yates and Martin W. Hetzer applied proteomics to examine protein turnover in cells of the rat central nervous system. They found that extremely long-lived proteins associated with chromatin and the nuclear pore complex did not turn over, potentially exposing these proteins to harmful metabolites and accumulation of damage over time. Read more about their findings in the journal Science to learn more. [Read Article]
News & Highlights: YRC Researchers Michael MacCoss and William Stafford Noble have published a new algorithm, dubbed Barista, for identifying proteins in complex biological mixtures. Instead of subdividing the task into separate peptide and protein identification tasks, Barista applies a machine learning approach to identify proteins from source spectra as a single optimization problem. Read their publications in Mol. Cell Proteomics to learn more. [Read Article]
News & Highlights: YRC researcher Stan Fields has used protein mass spectrometry to identify 870 unique sites of ubiquitin attachment on 438 different proteins in the budding yeast Saccharomyces cerevisiae. The analysis was based on the increase in molecular mass of a tryptic peptide carrying two additional glycine residues from the ubiquitin moiety. Read his paper in Proteomics to learn more. [Read Article]
News & Highlights: Hutchinson-Gilford progeria syndrome (HGPS) is a rare and fatal disease characterized by premature aging. In their recent collaboration, YRC researcher John Yates and collaborator Juan Carlos Izpisua Belmonte found induced pluripotent stem cells from HGPS patients lacked molecular characteristics associated with the disease, which were restored upon differentiation. See their paper in Nature to learn more. [Read Article]
News & Highlights: YRC researcher Stan Fields used the model organism Saccharomyces cerevisiae to probe the effects of nutritionally acquired metabolites on statins, a cholesterol-lowering drug widely prescribed to prevent heart disease. He found that copper and zinc ions impair the effect of statins by upregulating genes related to sterol production. Please read his paper in Molecular BioSystems to learn more. [Read Article]
News & Highlights: YRC researchers David Baker and Stan Fields have developed new technology for examining how a protein's sequence affects its function. This new technology is large-scale and may be applied to many in vitro or in vivo protein assays, providing a general means for studying the functional consequences of protein variation. Please read their paper in Nature Methods to learn more. [Read Article]
News & Highlights: The YRC collaborated with Sue Biggins at the Fred Hutchinson Cancer Research Center in Seattle to examine centromeres, whose proper function is critical to prevent conditions associated with cancer and some birth defects. This work, performed in yeast, was recently published in Molecular Cell, where Dr. Biggins proposes a new pathway for the regulation of centromeric function. [Read Article]
News & Highlights: Multidimensional protein identification technology (MudPIT) developed by the YRC was used in a recent collaboration with David Drubin at the University of California, Berkeley, to examine the assembly of actin networks in yeast. In his recent paper in Current Biology, Dr. Drubin describes the nucleation and assembly of these large protein complexes, and how MudPIT was used to characterize their composition. [Read Article]

YFP-CFP (G418) [pFA6a-YFP-CFP::KanR]

Plasmid pFA6a-YFP-CFP::KanR is similar to pDH18, except transformants are selected by G418 resistance. Forward and reverse primers to amplify the cassette are described on the protocol page "Fusing GFP to the Carboxy Terminus of Your Favorite Protein." This plasmid is provided by the Gould lab.

Map

Sequence

The DNA sequence of pFA6a-YFP-CFP::KanR below has the YFP sequence shaded green, the CFP sequence blue, and the primer sites red.
gaacgcggccgccagctgaagcttcgtacgctgcaggtcgacggatccccgggttaattaacatggtgagtaaaggaga agaacttttcactggagttgtcccaattcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtgga gagggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactggaaaactacctgttccatggc caacacttgtcactactttcggttatggtctaaaatgctttgctagatacccagatcatatgaaacggcatgacttttt caagagtgccatgcccgaaggttatgtacaggaaagaactatatttttcaaagatgacgggaactacaagacacgtgct gaagtcaagtttgaaggtgatacccttgttaatagaatcgagttaaaaggtattgattttaaagaagatggaaacattc ttggacacaaattggaatacaactataactcacacaatgtatacatcatggcagacaaacaaaagaatggaatcaaagt taacttcaaaattagacacaacattgaagatggaagcgttcaactagcagaccattatcaacaaaatactccaattggc gatggccctgtccttttaccagacaaccattacctgtcctatcaatctgccctttcgaaagatcccaacgaaaagagag accacatggtccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaaggcgccagtaa aggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgttaatgggcacaaattttctgtc agtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactggaaaactacctgttc catggccaacacttgtcactactttgacttggggtgttcaatgcttttcaagatacccagatcatatgaaacggcatga ctttttcaagagtgccatgcccgaaggttatgtacaggaaagaactatatttttcaaagatgacgggaactacaagaca cgtgctgaagtcaagtttgaaggtgatacccttgttaatagaatcgagttaaaaggtattgattttaaagaagatggaa acattcttggacacaaattggaatacaactatatttcacacaatgtatacatcactgcagacaaacaaaagaatggaat caaagctcatttcaaaattagacacaacattgaagatggaagcgttcaactagcagaccattatcaacaaaatactcca attggcgatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaaa agagagaccacatggtccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaataggg cgcgccacttctaaataagcgaatttcttatgatttatgatttttattattaaataagttataaaaaaaataagtgtat acaaattttaaagtgactcttaggttttaaaacgaaaattcttattcttgagtaactctttcctgtaggtcaggttgct ttctcaggtatagtatgaggtcgctcttattgaccacacctctaccggcagatccgctagggataacagggtaatatag atctgtttagcttgcctcgtccccgccgggtcacccggccagcgacatggaggcccagaataccctccttgacagtctt gacgtgcgcagctcaggggcatgatgtgactgtcgcccgtacatttagcccatacatccccatgtataatcatttgcat ccatacattttgatggccgcacggcgcgaagcaaaaattacggctcctcgctgcagacctgcgagcagggaaacgctcc cctcacagacgcgttgaattgtccccacgccgcgcccctgtagagaaatataaaaggttaggatttgccactgaggttc ttctttcatatacttccttttaaaatcttgctaggatacagttctcacatcacatccgaacataaacaaccatgggtaa ggaaaagactcacgtttcgaggccgcgattaaattccaacatggatgctgatttatatgggtataaatgggctcgcgat aatgtcgggcaatcaggtgcgacaatctatcgattgtatgggaagcccgatgcgccagagttgtttctgaaacatggca aaggtagcgttgccaatgatgttacagatgagatggtcagactaaactggctgacggaatttatgcctcttccgaccat caagcattttatccgtactcctgatgatgcatggttactcaccactgcgatccccggcaaaacagcattccaggtatta gaagaatatcctgattcaggtgaaaatattgttgatgcgctggcagtgttcctgcgccggttgcattcgattcctgttt gtaattgtccttttaacagcgatcgcgtatttcgtctcgctcaggcgcaatcacgaatgaataacggtttggttgatgc gagtgattttgatgacgagcgtaatggctggcctgttgaacaagtctggaaagaaatgcataagcttttgccattctca ccggattcagtcgtcactcatggtgatttctcacttgataaccttatttttgacgaggggaaattaataggttgtattg atgttggacgagtcggaatcgcagaccgataccaggatcttgccatcctatggaactgcctcggtgagttttctccttc attacagaaacggctttttcaaaaatatggtattgataatcctgatatgaataaattgcagtttcatttgatgctcgat gagtttttctaatcagtactgacaataaaaagattcttgttttcaagaacttgtcatttgtatagtttttttatattgt agttgttctattttaatcaaatgttagcgtgatttatattttttttcgcctcgacatcatctgcccagatgcgaagtta agtgcgcagaaagtaatatcatgcgtcaatcgtatgtgaatgctggtcgctatactgctgtcgattcgatactaacgcc gccatccagtttaaacgagctcgaattcatcgatgatatcagatccactagtggcctatgcggccgcggatctgccggt ctccctatagtgagtcgtattaatttcgataagccaggttaacctgcattaatgaatcggccaacgcgcggggagaggc ggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtat cagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggcca gcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaa aatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcg tgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctca atgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcag cccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcag ccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggcta cactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatcc ggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaag aagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatt atcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaact tggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcct gactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagaccc acgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaacttta tccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttg ttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaag gcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttg gccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctg tgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacg ggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaagg atcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcacca gcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatact catactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatt tagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatca tgacattaacctataaaaataggcgtatcacgaggccctttcgtctcgcgcgtttcggtgatgacggtgaaaacctctg acacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtca gcgggtgttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatggaca tattgtcgttagaacgcggctacaattaatacataaccttatgtatcatacacatacgatttaggtgacactata

Plasmids & Protocols