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Genetic engineering tinkers with genes

A new technique will help biologists alter genes. Whether to turn cells into tiny factories and churn out medicines, or modify crops to grow with limited water, or study the effects of a gene on human health, this technique will work on all life forms from bacteria to animals.


Messenger RNAs are long molecular chains made up of four kinds of links - adenine (A), cytosine (C), guanine (G) and uracil (U). Typically, these links are mixed together, like letters in a word. But, sometimes many A's occur in a row. Such a sequence of A's is slippery. The molecular machinery translating RNA into a protein has a tendency to stop and slip on a long track of A's before it reaches the end — reducing the amount of protein produced.

Sergej Djuranovic PhD, assistant professor of cell biology and physiology at Washington University School of Medicine in St. Louis, and the study's senior author leads the study. His graduate student Laura Arthur and colleagues found that thee slippery strings of A's could be used to regulate the amount of protein produced from a gene.


The more A's researchers added to the beginning or middle of a piece of messenger RNA, the less protein produced from that RNA.

By carefully controlling the length of A's on a string, or introducing different molecular links in certain positions along a string, they could produce exactly as much protein as they wanted.


Djuranovic and colleagues tested the technique in bacteria, protozoa, yeast, plants, fruit flies, mouse and human cells. It worked ­the same way in all of them. RNA translation is an ancient process occuring across all lifeforms.

"The great thing about this is how simple it is," Djuranovic explains. "In the past, if you wanted a mutation that knocked down [reduced] gene expression by, say 30 percent, it took years of work and a lot of luck to find a situation like that. Now we can do it in a few days."

The technique, published Jan. 20 in Nature Communications, allows scientists to precisely regulate how much protein is produced from a particular gene. The process is simple yet innovative and, so far, works in everything from bacteria to plants to human cells.


"Basically, this is a universal toolkit for modifying gene expression. It's a tool that can be used whether you are genetically engineering cells to produce a particular organic molecule, or to study how a gene works."

Sergej Djuranovic PhD, Assistant Professor, Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri, USA.


The ability to control the amount of protein produced from a particular gene would be a boon to biologists for designing or redesigning biological systems, such as a set of biochemical reactions to affect cell metabolism, or to produce a desired protein. For example, some drugs — including antibiotics such as vancomycin and cancer drugs such as taxol — are produced in cells as byproducts of metabolism. By fine-tuning certain genes, a biologist could maximize the quantity of medicine produced metabolically.


"There are all sorts of complex diseases such as cancer and autism in which we know expression from a particular gene is dialed down. But, nobody knows how that reduction is contributing to the disease.

"With classical genetics, you can only study what happens when you have two copies, one copy or no copies of a gene. In other words, 100 percent, 50 percent or zero percent gene expression. Now we can look at everything in between."

Sergej Djuranovic PhD


Djuranovic is interested in modifying gene expression to study disease-related genes, such as ones implicated in cancer.

Abstract
Hypomorphic mutations are a valuable tool for both genetic analysis of gene function and for synthetic biology applications. However, current methods to generate hypomorphic mutations are limited to a specific organism, change gene expression unpredictably, or depend on changes in spatial-temporal expression of the targeted gene. Here we present a simple and predictable method to generate hypomorphic mutations in model organisms by targeting translation elongation. Adding consecutive adenosine nucleotides, so-called polyA tracks, to the gene coding sequence of interest will decrease translation elongation efficiency, and in all tested cell cultures and model organisms, this decreases mRNA stability and protein expression. We show that protein expression is adjustable independent of promoter strength and can be further modulated by changing sequence features of the polyA tracks. These characteristics make this method highly predictable and tractable for generation of programmable allelic series with a range of expression levels.
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Jan 26, 2017   Fetal Timeline   Maternal Timeline   News   News Archive   



(a) Diagram of Fruit Fly larva: Salivary Glands (SG, BLUE), Central Nervous System (CNS, GREEN) and ProVentriculus (PV, RED). Expressed in 5 fly types by ROW: 1. Wild Type (WT), 2. (AAA)6 3. (AAA)9, 4. (AAA)-12, and 5. (AAG)12. By COLUMN: (b) SALIVARY GLAND: SG, (c) CENTRAL NERVOUS SYSTEM CNS and (d) PROVENTRICULUS PV; (mCherry and GFP indicate fluorescence filter settings; third column is overlay of mCherry and GFP)
Image Credit: Washington University School of Medicine, St. Louis, Missouri,

 


 


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