dna lyrics Archives - Gen9 Genetics
Introduction. A new type of selfish DNA element that was first identified in yeast in the 1970s (reviewed in Refs [54, 55]), self-targeting endonucleases have been highly successful in invading eukaryotic, bacterial, genomes. archeal and even phage despite its simple structure: a single open reading frame. Many (but not all) of the self-targeting endonucleases identified to date recognize highly specific DNA target sequences found in self-splicing introns. Others, known as inteins, are translated together with a host protein and processed post-translationally in a way that releases the self-targeted endonuclease but leaves the host protein intact. Both strategies have the advantage of limiting the detrimental effects on the host cell and its genome, allowing the propagation of these selfish genes. Following the identification and biochemical characterization of many self-directed endonucleases in the 1980s and 1990s, researchers began to reuse these simple elements for various genome manipulation applications. For example, the introduction of the target sequence recognized by endonuclease I-SceI into the Drosophila genome allowed homologous recombination and DNA break repair studies [56-59]. Self-directed endonucleases were relatively unknown to the vector biology community at this time, and their adaptation began when Burt  suggested that the primitive form of genome invasion used by self-directed endonucleases may be suitable for genetic strategies to control malaria. or other vector-borne diseases. diseases. Indeed, several self-directed endonucleases have since been used effectively to introduce site-specific DNA breaks in An. Gambiae [61,62] and to cleave genes from Ae. aegypti [63,64]. Through biochemical redesign, the self-directed endonucleases I-CreI and I-AniI have been modified to recognize targets in An. Gambiae genome directly . Transgenic expression of wild-type I-PpoI during spermatogenesis completely sterilized An. Gambiae mosquitoes ; Large cage trials using these sterile transgenic mosquitoes indicate their efficacy in crushing populations of this mosquito . The expression of modified versions of I-PpoI in the male testes of An. Gambiae has led to the development of sex-distorting strains that are produced by almost all males , while I-SceI was shown to successfully invade large populations of cages .
How they work. The phrase "homing" refers to their ability as mature proteins to return to their mRNA "birth" site and introduce a double-stranded break in the homologous chromosome lacking the homing endonuclease gene. Unlike ETs and recombinases, self-directed endonucleases do not catalyze any reaction beyond DNA cleavage. This extraordinarily simplistic mode of action therefore relies entirely on the cellular DNA repair machinery to generate a duplicate version of the local endonuclease gene, using the original gene as a repair template. Indigenous endonucleases can be classified by their way of catalyzing DNA cleavage into at least four completely independent families, and there is an extensive literature on the structural basis of DNA-binding specificity and double-stranded DNA break formation (revised in Ref. ). Of particular interest is that the target sequences for most self-directed endonucleases are 18 bp or more, which means that even in large eukaryotic genomes the probability of finding an endogenous site is very small. For example, the target sequence of I-SceI is 18 bp, and the corresponding probability of finding an exact match in a random sequence is 1 in 6.87 × 1010. Given the size of An. Gambiae (2.7 × 108 bp) and Ae. aegypti (1.3 × 109 bp), the corresponding chances of randomly finding a target I-SceI site are approximately 1: 250 and 1:50, respectively.
Strengths. Self-directed endonuclease genes are relatively small, with dimeric nucleases such as I-CreI and I-PpoI, both of less than 500 bp; the largest monomeric nucleases, such as I-SceI, are still less than 1 kb. Therefore, these genes and their resulting proteins are easy to manipulate, express, and purify. When integrated into the genome of a mosquito, its small size can also reduce the risks of acquiring deleterious mutations that can affect its function. Its extreme specificity can be expected to reduce or virtually eliminate the impacts of off-target logging; transgenic expression of I-SceI is well tolerated by both