Technique Developed To Count Messages Generated by Single Genes

The technology could provide a look into the process by which genes are turned on inappropriately, causing disease.

The new technique, devised by researchers from Albert Einstein College of Medicine of Yeshiva University (New York, NY, USA), provides a detailed window into mechanisms that until now were proven but never visualized. The more detailed view of DNA being made into RNA in a single cell will help answer questions about how much of a gene is made over time and how much that level varies from cell to cell. Insight into how genes work at a more precise level ultimately furthers understanding of disease processes that trigger cancer, for example, which arise when genes no longer work at their correct capacity or time.

"The classic textbook cartoon illustration of a single strand of DNA with little mRNA pieces coming off it can now be shown with real photographs,” explained Daniel Zenklusen, Ph.D., an Einstein post-doctoral fellow and first author of the study. The technique was developed in the laboratory of Robert Singer, Ph.D., cochair and professor of anatomy and structural biology at Einstein.

The new technology is a powerful modification of fluorescent in situ hybridization (FISH), developed in Dr. Singer's laboratory more than 26 years ago. FISH is now a widely used research application to study gene activation; that is, how much a gene has been activated in groups of cells. FISH is also used in genetic counseling to detect the presence of gene characteristics that diagnose conditions including Down's syndrome or Prader-Willi syndrome.

Recent developments in fluorescence, microscopy, and data analysis enabled the more powerful FISH application described in the study. Until this research, FISH could only be used to look at genes or their messages that are present at very high levels and only in tissues, not at the smaller level of the cell. However, this it the first time that all the individual mRNA molecules within single cells can be counted.

Dr. Singer's single RNA counting technique has the potential to change some fundamental hypotheses about how genes are regulated. As Dr. Singer explained, "our study using this new technique has already generated enough new ideas to keep students busy for the next 10 years.”

One of the most significant findings of this study was that "housekeeping” genes, which all cells need to survive, are not always expressed at a constant level. Variability, however, is restricted to a narrow range that seems to be characteristic for housekeeping genes. Combining single molecule measurement with mathematical modeling allowed the investigators to precisely determine how variability is controlled. This demonstrated that unlike the findings of previous studies, housekeeping genes are not transcribed by transcriptional bursts but at a rather constant rate. Bursting expression, however, is found in special classes of genes where higher variability might be an advantage for the cell. The next step is to see if this continuous/non-bursting theory of housekeeping gene control also applies to human cells. The research from Dr. Singer's team was performed in yeast cells.

Dr. Singer believes the approach of looking at biologic mechanisms in a natural environment rather than in a test tube at a single cell level reveals details that can advance the field of cancer and other disease research. "Cancer derives from a single cell. So current microarray technologies that are used on a tissue-wide level and are based on ‘grinding up a tumor' may be a good first step at directing us where to focus, but they may need to be combined with newer techniques that provide the precision to home in on single cells,” Dr. Singer said.

The study was published in the November 16, 2008, online edition of the journal Nature Structural and Molecular Biology.

Related Links:
Albert Einstein College of Medicine of Yeshiva University