导向进化和基质特异性概况的同源内核酶I-SceI
Jeffrey B Doyon1, Vikram Pattanayak, Carissa B Meyer
1Howard Hughes Medical Institute and the Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA.
Journal of the American Chemical Society
|February 16, 2006
概括
科学家们使用实验室进化工程设计了具有新的DNA切割能力的酶. 这一进步提供了新的基因组编辑工具和对核酶对DNA识别的更深入的见解.
科学领域:
- 分子生物学分子生物学
- 酵素工程是什么? 酶工程是什么
- 基因组学就是基因组学.
背景情况:
- 具有特定DNA裂变能力的酶对基因组操纵和理解DNA识别有价值.
- 像I-SceI这样的指导内核酶是识别和切割特定DNA序列的酶.
研究的目的:
- 开发和应用一种对具有量身定制的DNA裂变特征的进化酶的选择系统.
- 增强对核酶对特定序列DNA识别的理解.
- 为了设计突变的I-SceI定位内核酶,改变了DNA裂变的特异性.
主要方法:
- 开发一种高效的体内阳性和阴性选择系统,以施加进化压力.
- 在体外选择的应用,以确定野生类型I-SceI的基质特异性概况.
- 利用这些工具进化突变的I-SceI酶.
主要成果:
- 成功开发和应用一种用于酶进化的体内选择系统.
- 野生类型I-SceI基质特异性概况的表征.
- 突变I-SceI酶的进化,具有改变的DNA裂变特异性.
- 最进化的突变酶对目标突变DNA序列具有很高的选择性.
结论:
- 开发的选择系统对于具有新型DNA分裂特异性的进化酶是有效的.
- 工程酶可以达到与野生类型酶相当的DNA裂变选择性.
- 这些进展为基因组工程和研究DNA-蛋白相互作用提供了新的工具.
相关概念视频
In-vitro Mutagenesis
To learn more about the function of a gene, researchers can observe what happens when the gene is inactivated or “knocked out,” by creating genetically engineered knockout animals. Knockout mice have been particularly useful as models for human diseases such as cancer, Parkinson’s disease, and diabetes.
Non-LTR Retrotransposons
As the name suggests, non-LTR retrotransposons lack the long terminal repeats characteristic of the LTR retrotransposons. Additionally, both LTR and non-LTR retrotransposons use distinct mechanisms of mobilization. Non-LTR retrotransposons are further divided into two classes - Long interspersed nuclear elements (LINEs) and short interspersed nuclear elements (SINEs), both of which occur abundantly in most mammals, including humans. Some of the active non-LTR retrotransposons in humans are L1...
Conservative Site-specific Recombination and Phase Variation
Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP...
The recognition sites for Cre recombinase called LoxP...
Cis-regulatory Sequences
Cis-regulatory sequences are short fragments of non-coding DNA that are present on the same chromosomes as the genes that they regulate. These fragments serve as binding sites for transcriptional regulators, proteins that are responsible for controlling gene transcription and differential gene expression across cell types in eukaryotes. Cis-regulatory sequences can be close to the gene of interest or thousands of bases away in the DNA sequence; however, those sequences that are further away are...
Restriction Enzymes
Restriction enzymes are bacterial enzymes used to cut DNA in a sequence-specific manner. To cleave DNA, they bind to specific palindromic sequences called restriction sites. Such palindromic DNA sequences or inverted repeats are commonly found in regions of functional significance, such as the origin of replication, gene operator sites, and regions containing transcription termination signals.
The host bacteria protect their own genomic DNA from these enzymes by methylating these sites. Some...
The host bacteria protect their own genomic DNA from these enzymes by methylating these sites. Some...
Cis-regulatory Sequences
Cis-regulatory sequences are short fragments of non-coding DNA that are present on the same chromosomes as the genes that they regulate. These fragments serve as binding sites for transcriptional regulators, proteins that are responsible for controlling gene transcription and differential gene expression across cell types in eukaryotes. Cis-regulatory sequences can be close to the gene of interest or thousands of bases away in the DNA sequence; however, those sequences that are further away are...


