Quantitative imaging of TATA‐binding protein in living yeast cells
We describe the quantitative monitoring of TATA‐binding protein (TBP) localization and expression in living Saccharomyces cerevisiae cells. We replaced the endogenous TBP with a green fluorescent protein (GFP) · TBP fusion, which was imaged quantitatively by laser scanning confocal microscopy (LSCM)...
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| Veröffentlicht in: | Yeast (Chichester, England) England), 1998-06, Vol.14 (9), p.813-825 |
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| creator | Patterson, George H. Schroeder, Stephanie C. Bai, Yu Weil, P. Anthony Piston, David W. |
| description | We describe the quantitative monitoring of TATA‐binding protein (TBP) localization and expression in living Saccharomyces cerevisiae cells. We replaced the endogenous TBP with a green fluorescent protein (GFP) · TBP fusion, which was imaged quantitatively by laser scanning confocal microscopy (LSCM). When GFP · TBP expression was altered by using various promoters, the levels measured by LSCM correlated well with the levels determined by immunoblot of whole cell extract protein. These results show that GFP · TBP imaging not only offers a method of measurement equivalent to a more conventional technique but also provides real‐time quantitation in living cells and subcellular localization information. Time‐lapse confocal imaging of GFP · TBP in mitotic yeast cells revealed that it remains localized to the nucleus and displays an asymmetric distribution (1:0·7) between mother and daughter cells. Based on this and data from a mutant which underexpresses GFP · TBP, we suggest that intracellular levels of TBP are near rate‐limiting for growth and viability. © 1998 John Wiley & Sons, Ltd. |
| doi_str_mv | 10.1002/(SICI)1097-0061(19980630)14:9<813::AID-YEA280>3.0.CO;2-2 |
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Time‐lapse confocal imaging of GFP · TBP in mitotic yeast cells revealed that it remains localized to the nucleus and displays an asymmetric distribution (1:0·7) between mother and daughter cells. 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Anthony</creatorcontrib><creatorcontrib>Piston, David W.</creatorcontrib><title>Quantitative imaging of TATA‐binding protein in living yeast cells</title><title>Yeast (Chichester, England)</title><addtitle>Yeast</addtitle><description>We describe the quantitative monitoring of TATA‐binding protein (TBP) localization and expression in living Saccharomyces cerevisiae cells. We replaced the endogenous TBP with a green fluorescent protein (GFP) · TBP fusion, which was imaged quantitatively by laser scanning confocal microscopy (LSCM). When GFP · TBP expression was altered by using various promoters, the levels measured by LSCM correlated well with the levels determined by immunoblot of whole cell extract protein. These results show that GFP · TBP imaging not only offers a method of measurement equivalent to a more conventional technique but also provides real‐time quantitation in living cells and subcellular localization information. Time‐lapse confocal imaging of GFP · TBP in mitotic yeast cells revealed that it remains localized to the nucleus and displays an asymmetric distribution (1:0·7) between mother and daughter cells. Based on this and data from a mutant which underexpresses GFP · TBP, we suggest that intracellular levels of TBP are near rate‐limiting for growth and viability. © 1998 John Wiley & Sons, Ltd.</description><subject>Cell Nucleus - chemistry</subject><subject>DNA-Binding Proteins - analysis</subject><subject>DNA-Binding Proteins - genetics</subject><subject>DNA-Binding Proteins - metabolism</subject><subject>Gene Expression Regulation, Fungal</subject><subject>Green Fluorescent Proteins</subject><subject>Immunoblotting</subject><subject>Indicators and Reagents</subject><subject>Luminescent Proteins - metabolism</subject><subject>Microscopy, Confocal</subject><subject>Mitosis</subject><subject>Recombinant Fusion Proteins - analysis</subject><subject>Recombinant Fusion Proteins - genetics</subject><subject>Recombinant Fusion Proteins - metabolism</subject><subject>Saccharomyces cerevisiae</subject><subject>Saccharomyces cerevisiae - chemistry</subject><subject>Saccharomyces cerevisiae - genetics</subject><subject>Saccharomyces cerevisiae - growth & development</subject><subject>Subcellular Fractions - chemistry</subject><subject>TATA Box</subject><subject>TATA-Box Binding Protein</subject><subject>Time Factors</subject><subject>Transcription Factors - analysis</subject><subject>Transcription Factors - genetics</subject><subject>Transcription Factors - metabolism</subject><issn>0749-503X</issn><issn>1097-0061</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1998</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFUV1r2zAUFWOlS7v9hIGfRvvg7OrLkrIxZtyuCxTCWAbdXi6yLRcNx-kspyVv-wn7jf0llUnWlw0KAqF7j-459xxCPlKYUgD29uTrvJifUjAqBcjoCTVGQ8bhlIqZea8pn83y-Vn6_TxnGj7wKUyLxTuWsmdk8vjpOZmAEiaVwK9ekKMQfgJQKpk-JIdGU62omZCzLxvbDX6wg791iV_Za99dJ-smWebL_P73n9J39Vi56deD810ST-tvx8rW2TAklWvb8JIcNLYN7tX-PibfPp0vi8_p5eJiXuSXaSU0hVQ3jbSSm7qqRcmpKjMtjCllZl181CAyJpQWDVDegKqMkLqUqlGMSs1sVfFj8mY3N6r5tXFhwJUPowLbufUmoIprC5DZk0CaSRHZRuDVDlj16xB61-BNH03ot0gBxyQQxyRwNBVHU_FvEkgFGoxJIMYkcJcEcgQsFsiQxdGv9xo25crVj4P31sf-j13_zrdu-w_vk7T_Zd1X-ANJPqO4</recordid><startdate>19980630</startdate><enddate>19980630</enddate><creator>Patterson, George H.</creator><creator>Schroeder, Stephanie C.</creator><creator>Bai, Yu</creator><creator>Weil, P. 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Anthony ; Piston, David W.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c4810-8ff5a539dcd4b317b68499b56ae17bd04624784f013f07c9458b57f721582acc3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1998</creationdate><topic>Cell Nucleus - chemistry</topic><topic>DNA-Binding Proteins - analysis</topic><topic>DNA-Binding Proteins - genetics</topic><topic>DNA-Binding Proteins - metabolism</topic><topic>Gene Expression Regulation, Fungal</topic><topic>Green Fluorescent Proteins</topic><topic>Immunoblotting</topic><topic>Indicators and Reagents</topic><topic>Luminescent Proteins - metabolism</topic><topic>Microscopy, Confocal</topic><topic>Mitosis</topic><topic>Recombinant Fusion Proteins - analysis</topic><topic>Recombinant Fusion Proteins - genetics</topic><topic>Recombinant Fusion Proteins - metabolism</topic><topic>Saccharomyces cerevisiae</topic><topic>Saccharomyces cerevisiae - chemistry</topic><topic>Saccharomyces cerevisiae - genetics</topic><topic>Saccharomyces cerevisiae - growth & development</topic><topic>Subcellular Fractions - chemistry</topic><topic>TATA Box</topic><topic>TATA-Box Binding Protein</topic><topic>Time Factors</topic><topic>Transcription Factors - analysis</topic><topic>Transcription Factors - genetics</topic><topic>Transcription Factors - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Patterson, George H.</creatorcontrib><creatorcontrib>Schroeder, Stephanie C.</creatorcontrib><creatorcontrib>Bai, Yu</creatorcontrib><creatorcontrib>Weil, P. 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These results show that GFP · TBP imaging not only offers a method of measurement equivalent to a more conventional technique but also provides real‐time quantitation in living cells and subcellular localization information. Time‐lapse confocal imaging of GFP · TBP in mitotic yeast cells revealed that it remains localized to the nucleus and displays an asymmetric distribution (1:0·7) between mother and daughter cells. Based on this and data from a mutant which underexpresses GFP · TBP, we suggest that intracellular levels of TBP are near rate‐limiting for growth and viability. © 1998 John Wiley & Sons, Ltd.</abstract><cop>Chichester, UK</cop><pub>John Wiley & Sons, Ltd</pub><pmid>9818719</pmid><doi>10.1002/(SICI)1097-0061(19980630)14:9<813::AID-YEA280>3.0.CO;2-2</doi><tpages>13</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Cell Nucleus - chemistry DNA-Binding Proteins - analysis DNA-Binding Proteins - genetics DNA-Binding Proteins - metabolism Gene Expression Regulation, Fungal Green Fluorescent Proteins Immunoblotting Indicators and Reagents Luminescent Proteins - metabolism Microscopy, Confocal Mitosis Recombinant Fusion Proteins - analysis Recombinant Fusion Proteins - genetics Recombinant Fusion Proteins - metabolism Saccharomyces cerevisiae Saccharomyces cerevisiae - chemistry Saccharomyces cerevisiae - genetics Saccharomyces cerevisiae - growth & development Subcellular Fractions - chemistry TATA Box TATA-Box Binding Protein Time Factors Transcription Factors - analysis Transcription Factors - genetics Transcription Factors - metabolism |
| title | Quantitative imaging of TATA‐binding protein in living yeast cells |
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