Dr. Kelly Tatchell's Research Focus

Major Research Interests:
Regulation of cell growth and cell division; yeast genetics and cell biology.

A primary interest in our laboratory is the type 1 protein phosphatase (PP1) in the yeast Saccharomyces cerevisiae. This enzyme dephosphorylates phosphoserine and phosphothreonine residues on many proteins in vitro and has recently been shown to have a key regulatory role in physiological responses ranging from insulin-dependent activation of glycogen synthesis to the regulation of ion channels in the brain. It is an abundant enzyme in the nucleus and has a critical role in regulating in cell cycle. PP1 is evolutionarily conserved; yeast and human enzymes are over 80% identical. In yeast the enzyme is required for glycogen synthesis and is essential for cell division. The specificity of PP1 is thought to be determined by targeting subunits that regulate the activity of the phosphatase towards specific substrates and target the enzyme to specific subcellular compartments. We are using a combination of genetic and biochemical strategies to identify these regulatory subunits. We have recently identified subunits of PP1 that are required for such specific biological functions as glycogen biosynthesis, glucose repression, and cell cycle control. We have recently used video microscopy to locate PP1 in live cells that contain a Green Fluorescent Protein-PP1 hybrid gene. Originally from jellyfish, the Green Fluorescent Protein is a naturally fluorescent protein that can be expressed in a wide range of organisms. We have created a protein that contains both Green Fluorescent Protein and PP1 sequences. This fusion protein carries out all the essential functions of phosphatase and is fluorescent. Time-lapse video microscopy reveals that most of the PP1 is located in the nucleus, but significant quantities are located at other sites at specific times during the cell cycle. We are using this technology in combination with the genetics and biochemistry to determine how the subcellular distribution of PPA is controlled.

Protein Phosphatase 1		Green Fluorescent Protein

Plasmid pDH3

Plamid pDH3 contains the ORF for the cyan fluorescent protein, a variant of GFP with a blue shifted spectrum. The plasmid also contains the Kan selectable marker encoding resistance to the antibiotic G418. The codons have not been optimized for mammalian expression and instead are the same as in the parent vector (except for the 6 mutations listed below).

Source: Yeast Resource Center- University of Washington; Dr. Stan Fields

Map:

The DNA sequence of pDH3 (pFA6a-CFP-KAN):
GAACGCGGCCGCCAGCTGAAGCTTCGTACGCTGCAGGTCGACGGATCCCC GGGTTAATTAACAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAAT
TCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTG GAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATT TGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTT GACTTGGGGTGTTCAATGCTTTTCAAGATACCCAGATCATATGAAACGGC ATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACT ATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTT TGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTA AAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATATTTCA CACAATGTATACATCACTGCAGACAAACAAAAGAATGGAATCAAAGCTCA TTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACC ATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGAC AACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAA GAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACAC ATGGCATGGATGAACTATACAAATAGGGCGCGCCACTTCTAAATAAGCGA ATTTCTTATGATTTATGATTTTTATTATTAAATAAGTTATAAAAAAAATA AGTGTATACAAATTTTAAAGTGACTCTTAGGTTTTAAAACGAAAATTCTT ATTCTTGAGTAACTCTTTCCTGTAGGTCAGGTTGCTTTCTCAGGTATAGT ATGAGGTCGCTCTTATTGACCACACCTCTACCGGCAGATCCGCTAGGGAT AACAGGGTAATATAGATCTGTTTAGCTTGCCTCGTCCCCGCCGGGTCACC CGGCCAGCGACATGGAGGCCCAGAATACCCTCCTTGACAGTCTTGACGTG CGCAGCTCAGGGGCATGATGTGACTGTCGCCCGTACATTTAGCCCATACA TCCCCATGTATAATCATTTGCATCCATACATTTTGATGGCCGCACGGCGC GAAGCAAAAATTACGGCTCCTCGCTGCAGACCTGCGAGCAGGGAAACGCT CCCCTCACAGACGCGTTGAATTGTCCCCACGCCGCGCCCCTGTAGAGAAA TATAAAAGGTTAGGATTTGCCACTGAGGTTCTTCTTTCATATACTTCCTT TTAAAATCTTGCTAGGATACAGTTCTCACATCACATCCGAACATAAACAA CCATGGGTAAGGAAAAGACTCACGTTTCGAGGCCGCGATTAAATTCCAAC ATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCA ATCAGGTGCGACAATCTATCGATTGTATGGGAAGCCCGATGCGCCAGAGT TGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAG ATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAA GCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCC CCGGCAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAA AATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCC TGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGG CGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGAC GAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAGCT TTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTG ATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGA CGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTG CCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATG GTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGAT GAGTTTTTCTAATCAGTACTGACAATAAAAAGATTCTTGTTTTCAAGAAC TTGTCATTTGTATAGTTTTTTTATATTGTAGTTGTTCTATTTTAATCAAA TGTTAGCGTGATTTATATTTTTTTTCGCCTCGACATCATCTGCCCAGATG CGAAGTTAAGTGCGCAGAAAGTAATATCATGCGTCAATCGTATGTGAATG CTGGTCGCTATACTGCTGTCGATTCGATACTAACGCCGCCATCCAGTTTA AACGAGCTCGAATTCATCGATGATATCAGATCCACTAGTGGCCTATGCGG CCGCGGATCTGCCGGTCTCCCTATAGTGAGTCGTATTAATTTCGATAAGC CAGGTTAACCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTT GCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGG TCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGG TTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGG CCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCC ATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAG AGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGG AAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACC TGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGC TGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGT GCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATC GTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCC ACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTT CTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTA TCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCT TGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAA GCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCT TTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATT TTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTA AAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTG ACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTA TTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGAT ACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACC CACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGG GCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTAT TAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGC GCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTT GGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATG ATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCG TTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCA CTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGAC TGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGA GTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGA ACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTC AAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCAC CCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCA AAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAA ATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATC AGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAAT AAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGT CTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCA CGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGA CACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGG GAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGG GCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCAT ATGGACATATTGTCGTTAGAACGCGGCTACAATTAATACATAACCTTATGTATCATACACATACGATTTAGGTGACAC

Plasmid pDH5 contains the ORF for a pH tolerant yellow fluorescent protein, a variant of GFP. The plasmid also contains the selectable marker from S. pombe that complements S. cerevisiae his3. The codons have not been optimized for mammalian expression; instead the codons are the same as in the parent vector (except for the 5 mutations listed below).

Source: Yeast Resource Center- University of Washington; Dr. Stan Fields

Map:

Mutations in the GFP(S65T) ORF:

The DNA sequence of pDH5 (pFA6a-YFP-HIS):

GAACGCGGCCGCCAGCTGAAGCTTCGTACGCTGCAGGTCGACGGATCCC CGGGTTAATTAACAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCA ATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCA GTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATT TATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACT ACTTTCGGTTATGGTCTAAAATGCTTTGCTAGATACCCAGATCATATGA AACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGA AAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAA GTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTA TTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAA CTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGA ATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTC AACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGT CCTTTTACCAGACAACCATTACCTGTCCTATCAATCTGCCCTTTCGAAA GATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAG CTGCTGGGATTACACATGGCATGGATGAACTATACAAATAGGGCGCGCC ACTTCTAAATAAGCGAATTTCTTATGATTTATGATTTTTATTATTAAAT AAGTTATAAAAAAAATAAGTGTATACAAATTTTAAAGTGACTCTTAGGT TTTAAAACGAAAATTCTTATTCTTGAGTAACTCTTTCCTGTAGGTCAGG TTGCTTTCTCAGGTATAGTATGAGGTCGCTCTTATTGACCACACCTCTA CCGGCAGATCCGCTAGGGATAACAGGGTAATATAGATCTGTTTAGCTTG CCTCGTCCCCGCCGGGTCACCCGGCCAGCGACATGGAGGCCCAGAATAC CCTCCTTGACAGTCTTGACGTGCGCAGCTCAGGGGCATGATGTGACTGT CGCCCGTACATTTAGCCCATACATCCCCATGTATAATCATTTGCATCCA TACATTTTGATGGCCGCACGGCGCGAAGCAAAAATTACGGCTCCTCGCT GCAGACCTGCGAGCAGGGAAACGCTCCCCTCACAGACGCGTTGAATTGT CCCCACGCCGCGCCCCTGTAGAGAAATATAAAAGGTTAGGATTTGCCAC TGAGGTTCTTCTTTCATATACTTCCTTTTAAAATCTTGCTAGGATACAG TTCTCACATCACATCCGAACATAAACAACCATGGGTAGGAGGGCTTTTG TAGAAAGAAATACGAACGAAACGAAAATCAGCGTTGCCATCGCTTTGGA CAAAGCTCCCTTACCTGAAGAGTCGAATTTTATTGATGAACTTATAACT TCCAAGCATGCAAACCAAAAGGGAGAACAAGTAATCCAAGTAGACACGG GAATTGGATTCTTGGATCACATGTATCATGCACTGGCTAAACATGCAGG CTGGAGCTTACGACTTTACTCAAGAGGTGATTTAATCATCGATGATCAT CACACTGCAGAAGATACTGCTATTGCACTTGGTATTGCATTCAAGCAGG CTATGGGTAACTTTGCCGGCGTTAAAAGATTTGGACATGCTTATTGTCC ACTTGACGAAGCTCTTTCTAGAAGCGTAGTTGACTTGTCGGGACGGCCC TATGCTGTTATCGATTTGGGATTAAAGCGTGAAAAGGTTGGGGAATTGT CCTGTGAAATGATCCCTCACTTACTATATTCCTTTTCGGTAGCAGCTGG AATTACTTTGCATGTTACCTGCTTATATGGTAGTAATGACCATCATCGT GCTGAAAGCGCTTTTAAATCTCTGGCTGTTGCCATGCGCGCGGCTACTA GTCTTACTGGAAGTTCTGAAGTCCCAAGCACGAAGGGAGTGTTGTAAAG AGTACTGACAATAAAAAGATTCTTGTTTTCAAGAACTTGTCATTTGTAT AGTTTTTTTATATTGTAGTTGTTCTATTTTAATCAAATGTTAGCGTGAT TTATATTTTTTTTCGCCTCGACATCATCTGCCCAGATGCGAAGTTAAGT GCGCAGAAAGTAATATCATGCGTCAATCGTATGTGAATGCTGGTCGCTA TACTGCTGTCGATTCGATACTAACGCCGCCATCCAGTTTAAACGAGCTC GAATTCATCGATGATATCAGATCCACTAGTGGCCTATGCGGCCGCGGAT CTGCCGGTCTCCCTATAGTGAGTCGTATTAATTTCGATAAGCCAGGTTA ACCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTAT TGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTT CGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTAT CCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCA GCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCAT AGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGA GGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGG AAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATAC CTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCAC GCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTG TGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAAC TATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAG CAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTAC AGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTA TTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTG GTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTT TGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGAT CCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCAC GTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGAT CCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAG TAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCT CAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGT GTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCA ATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAA ACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATC CGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGT TCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCG TGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCA ACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTT AGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGT TATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCC ATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTC TGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATAC GGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGG AAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGA TCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTT TTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGC CGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTC TTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGA GCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCC GCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATT ATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTC TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCC GGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCC CGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACT ATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGGACATATT GTCGTTAGAACGCGGCTACAATTAATACATAACCTTATGTATCATACAC ATACGATTTAGGTGACACTATA