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The mantra of molecular biology – DNA makes RNA, which makes protein* – has pretty much ignored pseudogenes. Considered defective copies of DNA segments, the 20,000 pseudogenes in the human genome are thought to be non-participants in the protein-production assembly line.
Now, scientists in Japan and at the University of California, San Diego (UCSD) School of Medicine have discovered a novel regulatory role for one pseudogene, showing that it stabilizes a similar protein-coding gene on another chromosome. When the pseudogene was disabled, protein-production was compromised, with resulting abnormal kidneys and bones in laboratory mice. When a functioning pseudogene was re-introduced into mouse embryos, the mice developed normally.
Published in the May 1, 2003 issue of the journal Nature, the study was led by Shinji Hirotsune, M.D., Ph.D., Division of Neuro-Science, Research Center for Genomic Medicine, Saitama Medical School, Japan. Hirotsune collaborated with Anthony Wynshaw-Boris, M.D., Ph.D., UCSD associate professor of pediatrics and medicine, in whose lab he first starting exploring pseudogene function several years ago.
"These findings have implications for treating human disorders," said Hirotsune. "The mice get disease if the pseudogene is interrupted, so theoretically it's possible that a malfunctioning pseudogene may cause human disease, as well."
The team's discovery happened by chance. The researchers were making transgenic mice for a totally different experiment. The process included injection of DNA into fertilized eggs, causing the DNA to randomly integrate into the mouse genome.
" You hope that a transgenic mouse will manifest direct effects from the gene that you inject, so you can learn something about the function of that gene," said Wynshaw-Boris. "However, because the inserted gene randomly integrates itself, sometimes it fortuitously interrupts another gene that's in the genome and produces mice with characteristics you didn't expect. In this case, we noticed that one group of mice developed unusual and severe disease characteristics."
Almost all the mice in that group died. The few that survived had severe kidney and bone problems, which they passed on to their offspring. The researchers decided to pursue these unexpected findings.
"In this day and age, it's relatively straightforward to clone the sites where transgenes are integrated," Wynshaw-Boris continued. "In cloning this site, we found three genes near the location where the injected gene was integrated."
With further laboratory tests, the scientists ruled out two of the genes, and determined that the third gene – a pseudogene called makorin1-p1 – was responsible for abnormalities in the mice.
"Since a pseudogene does not have the ability to make a protein, we wanted to know how this pseudogene could cause the disease states in the mice," Hirotsune said.
The researchers noted that the pseudogene, makorin1-p1, is a fragmented gene similar to a full-fledged protein-coding gene called makorin1, which is located on a different chromosome.
In examinations of normal mouse kidneys, the protein-coding makorin1 was shown to be strongly expressed and highly visible. In mice with the disabled makorin1-p1 pseudogene, the expression of the makorin1 gene appeared weak and irregular in the diseased kidney tissue. Additional lab tests and experiments in mice determined that makorin1-p1 played a key role in regulating the stability of makorin1.
"We further confirmed that this regulation was occurring by correcting the defect in the line of the initial transgenic mice," Wynshaw-Boris said. "If the mouse embryo had a defective copy of the pseudogene, we replaced it with a normal copy, which produced a healthy mouse."
In continuing studies, the researchers hope to show the pseudogene-gene interaction is a general mechanism taking place in many cellular interactions.
Additional authors on the paper in Nature were Noriyuki Yoshida, Research Center for Genomic Medicine, Saitama Medical School, Japan; Amy Chen and Lisa Garrett, Genetic Disease Research Branch, National Human Genome Research Institute, National Institutes of Health; Fumihiro Sugiyama, Satoru Takahashi and Ken-ichi Yagami, Institute of Basic Medical Sciences and Laboratory Animal Resource Center, University of Tsukuba, Japan; and Atsushi Yoshiki, Experimental Animal Division, Department of Biological Systems, BioResource Center, RIKIN Tsukuba Institute, Japan.
Funding for the study came from PREST, Japan Science and Technology Corporation, and PROBRAIN.
* Located in the nucleus, double-stranded DNA carries the genetic information of a cell. Its thousands of genes act like instruction manuals to build proteins, which perform important tasks for cell functions or serve as the body's building blocks. RNA is an intermediate between DNA and protein. Parts of the DNA are "transcribed" into RNA, a single-stranded molecule, which contains the information needed to make the protein. This protein-coding RNA moves out of the nucleus, into the cytoplasm of the cell, and directs the assembly of amino acids that fold into a completed protein.
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File Date: 05.02.03
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