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Volumn 279, Issue 5347, 1998, Pages 95-98

Discrete start sites for DNA synthesis in the yeast ARS1 origin

Author keywords

[No Author keywords available]

Indexed keywords

FUNGAL DNA;

EID: 0032472223     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.279.5347.95     Document Type: Article
Times cited : (134)

References (33)
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    • 4; modified from C. Santocanale and J. F. X Diffley, EMBO J. 15, 6671 (1996)]. Thirty cycles of 1 min at 95°C, 1 min at 70°C, and 1.5 min at 72°C were performed in a Perkin-Elmer Thermocycler (Gene Amp PCR System 2400). Samples were extracted with chloroform, precipitated with ethanol, and resuspended in 3 μl of 95% formamide loading buffer. Samples were fractionated on 6% polyacrylamide-8 M urea sequencing gels. Primers used for RIP mapping were as follows: SV40/ 5016, 5′-CTTCATCTCCTCCTTTATCAGGATG-3′ (nt 5016 to 5040); SV40/154, 5′-CAGCAGGCAGAAGTATGCAAAGC-3′ (nt 154 to 131); rev IV, 5′-GCTTCCGGCTCGTATGTTGTGTGG-3′ (nt 617 to 640); and -90, 5′-CTGGCGAAAGGGGGATGTGCTG-3′ (nt 1032 to 1011). Nucleotide positions correspond to those in the maps shown in Fig. 3.
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    • A.-K. Bielinsky and S. A. Gerbi, data not shown
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    • note
    • -&WT by cloning a wild-type ARS1 copy into the Aat II restriction site of the shuttle vector pARS1/858-865 (3). The 193-bp wild-type copy of ARS1 (nt 734 to 926 shown in Fig. 3B) was amplified by annealing the oligonucleotides 5′-ATATATGACGTCACTCTAACAAAATAGCAAATTTC-3′ and 5′-ATATATGACGTCACAATCAATCAAAAAGCCAAA-3′, carrying an Aat Il restriction site, to pARS/WTA plasmid DNA and extending them with Taq polymerase. Both ARS1 copies (active and inactive) have the same orientation.
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    • note
    • We thank C. Liang and B. Stillman for advice and yeast ARS clones, E. A. Hendrickson for the CV-1 cell line and SV40, M. L. DePamphilis for discussion, A. Landy for critical reading of the manuscript, and E. A. Hendrickson, Z. Han, and members of our laboratory for fruitful discussions. Supported by NIH grant GM 35929.


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