TRE重组酶的分子进化

Biology

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Summary

在这里,我们报告TRE重组,通过定向,分子进化的一代。 TRE重组确认的HIV - 1病毒DNA的LTR序列内的一个预先定义的目标序列,切除和消灭前病毒从被感染的人体细胞。尽管还处于起步阶段,定向分子进化将允许创建自定义的酶,将作为分子的外科手术和分子医学工具。

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Buchholz, F. Molecular Evolution of the Tre Recombinase. J. Vis. Exp. (15), e791, doi:10.3791/791 (2008).

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Abstract

在这里,我们报告TRE重组,通过定向,分子进化的一代。 TRE重组确认的HIV - 1病毒DNA的LTR序列内的一个预先定义的目标序列,切除和消灭前病毒从被感染的人体细胞。

我们开始与CRE,一个38 kDa的重组,即承认一个34 bp的双链DNA序列loxP位知名。由于Cre重组可以有效地消除基因组序列,我们制定了一个可以去除5' - LTR和3' - LTR一个集成的HIV - 1病毒DNA序列之间的重组,裁缝。作为第一步,我们发现类似loxP位和重组活动测试的LTR网站内的序列。最初CRE和诱变的Cre库未能重组的HIV - 1病毒DNA的选择loxLTR网站。任何指示的分子进化过程的开始,至少需要剩余的活动,原来的非对称loxLTR序列分割成子集和测试再次重组活动。作为中间体,重组活动子集。通过反复的演进周期,接下来,重组图书馆丰富。随后,丰富图书馆的洗牌和重组。结合不同的突变,证明了协同和重组酶的重组loxLTR1和loxLTR2创建。这是证据,通过中间体的进化策略可以成功。共126演化周期后,个别重组酶功能和结构分析。最活跃的重组 - TRE - 创建19个氨基酸的改变。 TRE重组能够消费的HIV - 1前病毒基因组的HIV - 1感染的HeLa细胞(见“HIV - 1前病毒DNA切除使用一个演进的重组酶”,Hauber J.,海因里希Pette实验病毒学和免疫学研究所,德国汉堡)。虽然还处于起步阶段,定向分子进化将允许创建自定义的酶,将作为“分子手术”和分子医学工具。

Comments

3 Comments

  1. Dr. Frank Buchholz,

    Its nice to listen to the talk. I have some queries about Tre recombinase.,
     
    1, to excise a fragment using cre or tre i guess, there should be two loxp sites if you are targeting HIV LTR region it should be very specific region rather conserved region. So what happens if the LTR regions tend to mutate ?
    ², Isn't molecular evolution a long process to create such an recombinase ?

    3, There are "zinc finger nuclease" which are custom made enzyme which make specific cuts at DNA. It has the ability to bind to any piece of DNA. So will this technique be useful  for making "Zinc Finger Recombinase", which has the ability to bind LTR region and excise the whole or part of HIV genome.

    Hoping for your comments

    Roshan Padmanabhan
    India  

    Reply
    Posted by: Anonymous
    June 26, 2008 - 12:40 PM
  2. TRE can eradiction Lentiviral Vector from transducted cell fully? if Yes you can find different beatween Tat-Dependent and Tat-Independent LV as target for eradiction 5LTR-LTR3 from genome integrated sit? Sincerely

    Reply
    Posted by: Anonymous
    September 22, 2008 - 7:24 AM
  3. Hello Dr. Frank Bucholz,
    Have you sequenced the host cell genome in the wild type control vs. the HIV-1 infected vs Tre recombinase cells to
    definitively show where the HIV-1 genome is inserting, and that it is removed? Much evidence points to nonrandom insertion
    of the HIV-1 genome.
    Doug Cork
    MHRP/HJF (dcork@hivresearch.org)

    Reply
    Posted by: Anonymous
    October 21, 2009 - 10:06 AM

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