New tool for female reproductive genetics

May 29, 2018, Carnegie Institution for Science
The fruit fly Drosophila melanogaster is a powerful model organism for studying animal and human development and disease. Credit: Carnegie Institution for Science

The fruit fly Drosophila melanogaster is a powerful model organism for studying animal and human development and disease. It is low cost, generates rapidly, and there are many tools to genetically modify its cells. One tool is called the Gal4/UAS two-component activation system. It is a biochemical method used to study the process of turning a gene on (gene expression) and gene function. Although it has been a mainstay of Drosophila genetics for twenty-five years, it only functions effectively in non-reproductive cells, not in egg-producing cells. It has not been known why. Now, Carnegie's Steven DeLuca and Allan Spradling have discovered why and the have developed a new tool that can work in both cell types. The research is published in the June 2018 Genetics.

The Gal4 gene is a transcription factor. Transcription factors encode proteins that turn on. The Gal4 protein recognizes a so-called upstream activator sequence (UAS), which can induce the expression of a gene of interest. A special version of the UAS was made at the Department of Embryology in 1998, called UASp, to work during egg-cell development. But the fact that different tools are needed for non-reproductive cells and egg-forming cells has been a major limitation.

The original pUASt vector—a molecule that ferries foreign genetic material into another cell—contains a promotor called Hsp70. As the name suggests, promotors are bits of DNA that initiate or promote gene transcription. Researchers have developed several varieties to improve its expression. Hsp70 is a member of a family of proteins with similar structures in most all living organisms and are an important for protein folding and for protecting cells from stress. The mechanisms of protein folding are vital to life and to understanding diseases.

The variations of UAS, however, did not correct the major problem of poor genetic activity in the female egg-producing system compared with non-reproductive tissues. The main stumbling block to obtaining a widely effective GAL4 vector has been the lack of understanding why UASt functions poorly in egg-producing cells and the lack of research comparing UASp and UASt promoters.

DeLuca and Spradling studied the differences between the UASp and UASt promoters. Their research agreed with previous reports that UASt worked better than UASp in all non-reproductive tissues while UASp worked better in the female egg-producing system.

They also looked at the reason for the extremely weak UASt expression in the female reproductive system. The evidence indicated that non-coding RNA molecules (called piRNA ) orchestrated the silencing that limited UASt expression.

They then looked at where these UASt-piRNAs originated by testing to see if Hsp70 piRNAs were responsible for silencing. Their results strongly indicated that UASt is normally silenced by Hsp70 piRNAs and that UASt is better than UASp in cells lacking Hsp70 piRNAs.

"We next attempted to create a new version of the UAS expression vector that works well in both the non-reproductive and the egg-producing system, "remarked DeLuca. "We hypothesized that Hsp70 piRNAs might recognize UASt RNA to initiate piRNA silencing. To prevent Hsp70 piRNAs from recognizing UASt RNA, we trimmed down the UASt vector's nucleotides—the basic units of DNA and RNA—to be shorter than a single piRNA. We went from 213 nucleotides to 19 nucleotides. We named this shortened variant 'UASz,'because we hoped it would be the last one anyone would make!"

The scientists found that UASz was expressed about 4 times higher than UASp at all stages in the egg-producing system.

Spradling remarked, "UASz is a superior vector over UASp in all tissues, and it is equivalent to UASt in many, but not all, non-reproductive tissues. It is an unequivocal upgrade for all applications. This is a major hurdle overcome for reproductive studies. We hope it will unlock the floodgates of research in this area and others."

Explore further: DNA protection, inch by inch

Related Stories

DNA protection, inch by inch

July 9, 2015

DNA within reproductive cells is protected through a clever system of find and destroy: new research published in Cell Reports today lifts the veil on how this is done.

How cells hack their own genes

August 24, 2017

DNA in all organisms from yeast to humans encodes the genes that make it possible to live and reproduce. But these beneficial genes make up only 2 percent of our DNA. In fact, more than two-thirds of our genome is populated ...

How male germ cells avoid genome instability

March 28, 2018

A group of researchers at Osaka University reported the function of GTSF1 in male germ cells. The study, which can be read in EMBO Reports, shows that GTSF1 is an essential factor for secondary piRNA biogenesis by regulating ...

Chaperones just prepare proteins for folding on their own

April 26, 2018

Cellular proteins are produced as long chains of amino acids that must fold precisely into their final shape. The key players in this folding process are the so-called molecular chaperones, protein helpers that make sure ...

Recommended for you

What happened before the Big Bang?

March 26, 2019

A team of scientists has proposed a powerful new test for inflation, the theory that the universe dramatically expanded in size in a fleeting fraction of a second right after the Big Bang. Their goal is to give insight into ...

Cellular microRNA detection with miRacles

March 26, 2019

MicroRNAs (miRNAs) are short noncoding regulatory RNAs that can repress gene expression post-transcriptionally and are therefore increasingly used as biomarkers of disease. Detecting miRNAs can be arduous and expensive as ...

Race at the edge of the sun: Ions are faster than atoms

March 26, 2019

Scientists at the University of Göttingen, the Institut d'Astrophysique in Paris and the Istituto Ricerche Solari Locarno have observed that ions move faster than atoms in the gas streams of a solar prominence. The results ...

Physicists discover new class of pentaquarks

March 26, 2019

Tomasz Skwarnicki, professor of physics in the College of Arts and Sciences at Syracuse University, has uncovered new information about a class of particles called pentaquarks. His findings could lead to a new understanding ...

0 comments

Please sign in to add a comment. Registration is free, and takes less than a minute. Read more

Click here to reset your password.
Sign in to get notified via email when new comments are made.