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Volumn 287, Issue 5460, 2000, Pages 2026-2029

A role for nuclear inositol 1,4,5-trisphosphate kinase in transcriptional control

Author keywords

[No Author keywords available]

Indexed keywords

GENE PRODUCT; INOSITOL TRISPHOSPHATE 3 KINASE; MESSENGER RNA; PHOSPHOLIPASE C;

EID: 0034677903     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.287.5460.2026     Document Type: Article
Times cited : (359)

References (47)
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    • 5x were detected in these strains, indicating that other IP kinases may be present in yeast cells.
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    • Genomic sequence encoding ARG82 was isolated using Tag polymerase with yeast genomic DNA as template and oligonucleotide primers containing Bam HI restriction sites. Primers were designed based on yeast genomic DNA sequence (DDBJ/EMBL/GenBank). The resulting fragment was cloned into the Bam HI site of plasmids pRS315 and pRS316. These were transformed into strain SWY1852 (MATα gle3-3 gle1-2 ade2 ade3 ura3 his3 leu2 trp1 pGLE1/URA3/ADE3/CEN). Rescue of synthetic lethality was tested by monitoring for red and white sectoring of a gsl3-3 gle1-2 pGLE1/URA3/ADE3/CEN plPK2/LEU2/CEN strain on yeast extract, peptone, and dextrose (YPD) medium. The same strain was tested for temperature-sensitive growth at 37°C on medium lacking uracil (-Ura). Whereas a pLEU2/CEN plasmid does not confer sectoring on YPD or growth at 37°C on -Ura, the piPK2/LEU2/CEN plasmid resulted in sectoring and growth at 37°C. Soluble IP levels were analyzed as described above for the ipk2Δ strains. Because the haploid ipk2Δ strains are defective for mating (8), the ipk2Δ strain was transformed with pIPK2/URA3/CEN. The resulting haploid was mated with SWY 1852 (plus pRS315), and the diploid was assayed for temperature sensitivity after growth on 5-fluoroorotic acid.
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    • The amino-terminal end of IPK2 was fused in frame to CFP and expressed from a centromeric TRP1 plasmid under control of the CDC42 promoter in an ipk2Δ strain (26). Cells were grown to logarithmic growth phase in complete minimal medium minus tryptophan in the presence of 1 μg/ml DAPI. Cells were transferred to slides and viewed directly in growth medium using a Zeiss Axioskop microscope (Zeiss. Thornwood, NY) equipped with epiflorescent and differential interference contrast (DIC) optics. The tagged protein was functionally similarly to native Ipk2. Expression of GFP-tagged Ipk2 on a centromeric plasmid (with the CDC42 promoter) rescues the temperature sensitivity at 37°C, growth on ornithine, and defects in inositol phosphate production (as determined by steady-state labeling and HPLC analysis) of the ipk2Δ strain.
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    • 2, 5 μM L-arginine, 0.5 μg/ml herring sperm DNA, and 5% glycerol; complexes were resolved by electrophoresis on 5% nondenaturing polyacrylamide gels and visualized by autoradiography.
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    • note
    • Supported by the Duke University Medical Scientist Training Program (A.R.O.), grants from the American Cancer Society and the National Institutes of General Medical Sciences (S.R.W.), a Burroughs Wellcome Fund Career Award in the Biomedical Sciences (J.D.Y.), and a Whitehead Scholar Award (J.D.Y.).


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