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发布于:2019-3-14 00:02:47  访问:3 次 回复:0 篇
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1 -associated phenotypes. These transcription factors {include
These transcription aspects incorporate proteins involved in basal transcription
1 -associated phenotypes. These transcription components involve proteins involved in basal transcription including Rpb2p and Sua7p (yTFIIB) (13); transcription mediators including Sin4p (H.-Y. Fan and H. L. Klein, unpublished information), Srb2p and Hrs1p (32, 36), and genespecific activators for instance Gcr3p (44). A current search for high-copy-number suppressors of hpr1 uncovered PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/24247322 a novel gene, THO2/RLR1, that is involved in RNA polymerase II transcription (33, 50). The hyperrecombination phenotype linked with hpr1 mutants was also discovered to become far more serious when direct repeats have been highly transcribed (13), and recent evidence indicates that Hpr1p influences transcription elongation (7, 34). To further discover the connection involving Hpr1p and transcription, we‘ve studied a novel class of transcription components in which mutations are also hyperrecombinogenic and are le* Corresponding author. Mailing address: Division of Biochemistry, NYU Medical Center, 550 First Ave., New York, NY 10016. Phone: (212) 263-5778. Fax: (212) 263-8166. E-mail: M2951 mechanism of action hannah.klein @med.nyu.edu. Present address: Department of Molecular Biology, Massachusetts Common Hospital, Boston, MA 02114.FAN ET AL.MOL. CELL. BIOL.fragment containing the YDL085w sequence. pHF81-4, pHF127-3, and pHF80-4 have been buy TPPU generated by subcloning the SalI-SacI fragment of pHF68-1 into pRS315, YCplac111, and YEp351, respectively. To confirm that no adjustments had been introduced into the SUB2 gene through PCR amplification, the SUB2 insert in pHF80-4 was entirely sequenced and compared to the reported SUB2 sequence in the database. No modifications had been located. sub2 conditional alleles along with a wild-type handle SUB2 allele around the pRS315 vector (CEN6 LEU2) were kindly offered by Christine Guthrie and Amy Kistler. YEp-MUD2 was constructed by insertion on the MUD2 sequence into the high-copy-number plasmid YEplac112-TRP1. Primers five CGCGGATCCATAG AACCGCTCCCCATGTC3 and 5 GCGGGATCCGTCCTTCCATGAAGTTT GCCC3 have been made use of to amplify the MUD2 coding sequence. The PCR item was digested with BamHI and inserted in to the BamHI website of YEplac112. The mud2::URA3 deletion was created by the one-step gene disruption approach (35). PCR amplification was employed to make the deletion cassette. Each primer consisted of 40 bp in the five end that have been homologous to MUD2 followed by 20 bp that were homologous to URA3. The primers utilized have been 5 TATAGGAAAATC AGAAAAGGATGTTGTGCCGATTGAGAACAAAAGATTCATTGTACTG AGAGTGCACCAC3 and five TCGTCCTCATCTATATAAGTACACAGAAC AGTGCGATCGTTGAATTGCGTTGTGCGGTATTTCACACCGC3 . The mud2 deletion retained the first 100 bp as well as the final 84 bp from the MUD2 coding sequence. C terminus FLAG-tagged Sub2 protein was generated in vivo employing a PCRgenerated copy of SUB2 inserted in to the high-copy-number vector YEp351. SUB2 was amplified in the yeast genome (Expand Extended Template PCR Method; Roche Diagnostics) making use of primers 5 GAAGGGATTCCTCCGTGTA G3 and 5 CGCGGATCCTTACTTGTCATCGTCGTCCTTGTAGTCATTAT TCAAATAAGTGGACGG3 . The latter primer consists of the information for the FLAG epitope as well as a BamHI PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/25486018 restriction website. This construct was then cloned into YEp351 in the SalI and BamHI restriction sites. Four hundred seventy base pairs directly 3 of genomic SUB2 had been then PCR amplified (primers employed have been 5 CGCGGATCCAAAAAAGATACGTTTTTATATAG3 and five CGCGAGCTCCGAATTGAAGAAGGCCTTCACC3 ) and cloned in to the SUB2-FLAG-containing vector making use of the BamHI and SacI restriction web sites.1 -associated phenotypes.
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