DNA replication fork pause sites dependent on transcription

Science

Volumn 272, Issue 5264, 1996, Pages 1030-1033

  Deshpande, Atul M ^a,b   Newlon, Carol S ^a  

^a RUTGERS NEW JERSEY MEDICAL SCHOOL   (United States)

^b INDIANA UNIVERSITY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

RNA POLYMERASE; TRANSFER RNA;

ARTICLE; DNA REPLICATION; DNA TRANSCRIPTION; FUNGAL GENETICS; GENE MAPPING; GENE MUTATION; NONHUMAN; PRIORITY JOURNAL; PROTEIN ASSEMBLY; RNA TRANSCRIPTION; SACCHAROMYCES CEREVISIAE; TRANSCRIPTION INITIATION;

EID: 0029740114     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.272.5264.1030     Document Type: Article

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17. The 240-bp, 212-bp, 187-bp, and 159-bp fragments containing SUP53 or SUP53-a flanked by different amounts of sequences upstream of the transcription start site and downstream of the transcription termination site were amplified by polymerase chain reaction from plasmid pAT20 or YEP13-SUP53 (obtained from D. Engelke) Primers A (5′-CGCTGGATCCTCCTTGTTCATGTGTGTTC-3′) and B (5′-CGCTGGATCCTTTCTCAACAAGTAATTGG-3′) became annealed 60 bp and 7 bp upstream of the transcription start site, respectively. Primers C (5′-CGCTGGATCCTTGATTCTGTGCGATAGCG-3′) and D (5′-CGCTGGATCCGTTCTCGTTATGTTGAGG-3′) became annealed 46 bp and 18 bp downstream of the transcription termination site, respectively. Primers were designed to create Bam HI site at the ends (boldface letters). Amplified fragments were then cloned in both orientations in pAT1 (21).
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33. YNN214 is MATa, ura3-52, lys2-801, ade2-101 [R. Sikorski and P. Hieter, Genetics 122, 19 (1989)]. YAΔ306 is YNN214 carrying a deletion of ARS306 constructed as described (20). YP528 is YNN214 carrying a T to G point mutation at position 9 of the ARS consensus sequence of ARS307 (20). YP306D is MATα, ura3-52, lys2-801, ade2-101 carrying a deletion of ARS306 constructed as described (20). YNN281 is MATa, ura3-52, lys2-801, ade2-101 his3-Δ200, trp1-901. The ts41 strain is YNN281 carrying the rpc160-41 mutation (13). Both YNN281 and ts41 were transformed with plasmid pAT21B (Table 1) to create YNN281 -21B and ts41-21B, respectively. Media used were as described [F. Sherman, Methods Enzymol. 194, 3 (1991)].
34. YNN214 is MATa, ura3-52, lys2-801, ade2-101 [R. Sikorski and P. Hieter, Genetics 122, 19 (1989)]. YAΔ306 is YNN214 carrying a deletion of ARS306 constructed as described (20). YP528 is YNN214 carrying a T to G point mutation at position 9 of the ARS consensus sequence of ARS307 (20). YP306D is MATα, ura3-52, lys2-801, ade2-101 carrying a deletion of ARS306 constructed as described (20). YNN281 is MATa, ura3-52, lys2-801, ade2-101 his3-Δ200, trp1-901. The ts41 strain is YNN281 carrying the rpc160-41 mutation (13). Both YNN281 and ts41 were transformed with plasmid pAT21B (Table 1) to create YNN281 -21B and ts41-21B, respectively. Media used were as described [F. Sherman, Methods Enzymol. 194, 3 (1991)].
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36. pAT1 was constructed by inserting the 1.45-kb Eco RI fragment containing TRP1 and ARS1 in the Eco RI site of pVHA [J. V. Van Houten and C. S. Newlon, Mol. Cell. Biol 10, 3917 (1990)]. Fragments of interest were cloned at the Bam HI site of pAT1, and the plasmids were used to transform strain YNN214 (79) as described (20).
37. J. F. Theis and C. S. Newlon, Yeast 8, 223 (1992).
38. The 1-kb Eco RI-Bam HI fragments containing the wild-type SUP2 tRNA gene and the mutant sup2 tRNA gene containing the C56G transversion mutation in box B were isolated from pDLC365 and pDLC565, respectively, which were obtained from S. Sandmeyer.
39. We thank B. Brewer, L. Ford, D. Kaback, M. Newlon, J. Wilusz, and members of the Newlon laboratory for comments on the manuscript; and D. Engelke and S. Sandmeyer for supplying plasmids and strains. Supported by NIH grant GM35679 (C.S.N.) and in part by a graduate fellowship from UMDNJ-Graduafe School of Biomedical Science (A.M.D.).