Abstract:
:The design of a family shuffling strategy (CLERY: Combinatorial Libraries Enhanced by Recombination in Yeast) associating PCR-based and in vivo recombination and expression in yeast is described. This strategy was tested using human cytochrome P450 CYP1A1 and CYP1A2 as templates, which share 74% nucleotide sequence identity. Construction of highly shuffled libraries of mosaic structures and reduction of parental gene contamination were two major goals. Library characterization involved multiprobe hybridization on DNA macro-arrays. The statistical analysis of randomly selected clones revealed a high proportion of chimeric genes (86%) and a homogeneous representation of the parental contribution among the sequences (55.8 +/- 2.5% for parental sequence 1A2). A microtiter plate screening system was designed to achieve colorimetric detection of polycyclic hydrocarbon hydroxylation by transformed yeast cells. Full sequences of five randomly picked and five functionally selected clones were analyzed. Results confirmed the shuffling efficiency and allowed calculation of the average length of sequence exchange and mutation rates. The efficient and statistically representative generation of mosaic structures by this type of family shuffling in a yeast expression system constitutes a novel and promising tool for structure-function studies and tuning enzymatic activities of multicomponent eucaryote complexes involving non-soluble enzymes.
journal_name
Nucleic Acids Resjournal_title
Nucleic acids researchauthors
Abécassis V,Pompon D,Truan Gdoi
10.1093/nar/28.20.e88keywords:
subject
Has Abstractpub_date
2000-10-15 00:00:00pages
E88issue
20eissn
0305-1048issn
1362-4962journal_volume
28pub_type
杂志文章abstract::The nucleotide sequence of the Anacystis nidulans 23S rRNA gene, including the 5'- and 3'-flanking regions has been determined. The gene is 2876 nucleotides long and shows higher primary sequence homology to the 23S rRNAs of plastids (84.5%) than to that of E. coli (79%). The predicted rRNA transcript also shares many...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/12.7.3373
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abstract::The proportion of cytosines methylated in the DNA of nucleosome oligomers and of core particles appears indistinguishable from that of total nuclear DNA from CHO cells. However the DNA in nucleoprotein which is initially released from nuclei by treatment with very low levels of micrococcal nuclease and the first 10% o...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/4.9.3097
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journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/11.22.7769
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journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/23.15.3050
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journal_title:Nucleic acids research
pub_type: 杂志文章
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journal_title:Nucleic acids research
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journal_title:Nucleic acids research
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doi:10.1093/nar/gky461
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journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/12.1part2.521
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abstract::A sixty base pair DNA duplex containing the nucleotide sequence of the bacteriophage T5 early (T5P25) promoter has been constructed using a combination of chemical synthesis and enzymatic methods. Subsequent to cloning into pBR322, the promoter has been demonstrated to be biologically active being capable of directing...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/11.17.5921
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journal_title:Nucleic acids research
pub_type: 杂志文章
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更新日期:1993-06-25 00:00:00
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journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkm244
更新日期:2007-01-01 00:00:00
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journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gku303
更新日期:2014-06-01 00:00:00
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journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/3.1.231
更新日期:1976-01-01 00:00:00
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journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkq1238
更新日期:2011-01-01 00:00:00
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journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkt728
更新日期:2013-11-01 00:00:00
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journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkh598
更新日期:2004-05-11 00:00:00
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journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkg161
更新日期:2003-01-15 00:00:00
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journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/25.22.4513
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journal_title:Nucleic acids research
pub_type: 杂志文章
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journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/9.11.2455
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journal_title:Nucleic acids research
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journal_title:Nucleic acids research
pub_type: 杂志文章
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journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gku1216
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journal_title:Nucleic acids research
pub_type: 杂志文章
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journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkl559
更新日期:2006-01-01 00:00:00
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journal_title:Nucleic acids research
pub_type: 杂志文章
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更新日期:2014-02-01 00:00:00
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journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/6.5.1979
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journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/1.11.1411
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journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/12.9.4011
更新日期:1984-05-11 00:00:00