Forum for Science, Industry and Business

Sponsored by:     3M 
Search our Site:

 

New Technique Yields More Detailed Picture Of Chromatin Structure

17.04.2008
University of Illinois researchers have developed a technique for imaging cells under an electron microscope that yields a sharper image of the structure of chromatin, the tightly wound bundle of genetic material and proteins that makes up the chromosomes.

Their findings appear in Nature Methods.

Scientists have known for more than a century that proteins, such as histones, aid in packing DNA into the nucleus of a cell. Human cells contain 2 to 3 meters of DNA, which must be kinked and coiled enough to fit into a region 1/10 the width of a human hair.

Despite the use of powerful, high-resolution imaging techniques such as electron microscopy, the mechanism by which this chromatin packing occurs remains a mystery. The densely coiled chromatin fibers are very difficult to visualize, and little is known about how they condense during cell division, or unwind to allow gene expression.

In developing their method, the Illinois team tackled a key difficulty in imaging cells using electron microscopy. Traditional studies “fix” the cells with potent chemicals (called fixatives) to preserve their structure for viewing under a microscope. But standard fixation methods interfere with another step in the imaging process: the use of tagged antibodies to label key components of the cells.

These antibodies, which target and latch on to specific proteins in the cell, can be tagged with fluorescent labels for detection in light microscopy, or with metal particles (gold, in this case) for electron microscopy.

“If you fix the cells first, you have a dramatic drop in the efficiency of these immunochemical reactions,” said Igor Kireev, a visiting scientist in the department of cell and developmental biology and lead author of the paper.

electron microscopy image

“And if your target is inside the condensed chromatin, the antibodies have no way to penetrate.”

Instead of fixing the cells before staining with antibodies, the researchers first exposed living animal cells to the labeled antibodies. This allowed the antibodies to penetrate more deeply into the chromatin structure, and boosted the number of gold particles adhering to regions of interest. The signal was enhanced by adding a silver solution that precipitated (solidified) upon contact with the gold.

“We are interested in chromatin structure, so our targets are mostly chromatin-bound proteins,” Kireev said.

The researchers had inserted several copies of a bacterial DNA, called the Lac operator, into the chromosomes. A bacterial protein, the Lac repressor, recognizes and binds to the Lac operator in living cells.

The researchers combined a Lac repressor protein with another protein that fluoresces green under blue light. This engineered protein adhered to the chromosomes in regions containing the Lac operator sequences. Under blue light, these regions fluoresced. A gold-tagged antibody targeted against green fluorescent protein (GFP) was then microinjected into the nucleus of a living cell, which added a metallic signal that could be boosted with silver.

“All this combined gives us a much better signal, a much stronger signal, with the very best structural preservation,” Kireev said.

The fluorescing protein helped the researchers find the regions of interest in the cells. These areas were then “immunogold” labeled and targeted for electron microscopy.

In the resulting micrographs the researchers saw enhanced staining of the chromosomes.

“We can now apply this same live-cell labeling method to study at high resolution many different GFP-tagged proteins in the cell cytoplasm or nucleus,” said Andrew Belmont, a professor of cell and developmental biology and senior author of the paper.

“In trying to understand chromosomes, people have largely been limited to low resolution visualization of specific chromosomal proteins using light microscopy,” Belmost said. “This meant everyone has had to do a lot of guessing of how things are put together, leading in many cases to vague, cartoon models of what are likely to be complicated chromosomal structures carrying out DNA functions such as replication and transcription.”

“Now we hope we can simply look and see the real structure using the more than 10-fold higher resolution of electron microscopy,” Belmont said. “We are really excited to see what we will find using our new method”

Editor’s note: To reach Andrew Belmont, call 217-244-2311; e-mail: asbel@uiuc.edu.

To reach Igor Kireev, call 217-333-8372; e-mail: ikireev@uiuc.edu.

Diana Yates | University of Illinois
Further information:
http://www.uiuc.edu
http://www.news.uiuc.edu/news/08/0416immunogold.html

Further reports about: Antibodies Chromatin DNA Electron Kireev Lac Microscopy chromosomes

More articles from Life Sciences:

nachricht Research team of the HAW Hamburg reanimated ancestral microbe from the depth of the earth
01.03.2017 | Hochschule für Angewandte Wissenschaften Hamburg

nachricht Researchers Imitate Molecular Crowding in Cells
01.03.2017 | Universität Basel

All articles from Life Sciences >>>

The most recent press releases about innovation >>>

Die letzten 5 Focus-News des innovations-reports im Überblick:

Im Focus: Researchers Imitate Molecular Crowding in Cells

Enzymes behave differently in a test tube compared with the molecular scrum of a living cell. Chemists from the University of Basel have now been able to simulate these confined natural conditions in artificial vesicles for the first time. As reported in the academic journal Small, the results are offering better insight into the development of nanoreactors and artificial organelles.

Enzymes behave differently in a test tube compared with the molecular scrum of a living cell. Chemists from the University of Basel have now been able to...

Im Focus: Safe glide at total engine failure with ELA-inside

On January 15, 2009, Chesley B. Sullenberger was celebrated world-wide: after the two engines had failed due to bird strike, he and his flight crew succeeded after a glide flight with an Airbus A320 in ditching on the Hudson River. All 155 people on board were saved.

On January 15, 2009, Chesley B. Sullenberger was celebrated world-wide: after the two engines had failed due to bird strike, he and his flight crew succeeded...

Im Focus: Breakthrough with a chain of gold atoms

In the field of nanoscience, an international team of physicists with participants from Konstanz has achieved a breakthrough in understanding heat transport

In the field of nanoscience, an international team of physicists with participants from Konstanz has achieved a breakthrough in understanding heat transport

Im Focus: DNA repair: a new letter in the cell alphabet

Results reveal how discoveries may be hidden in scientific “blind spots”

Cells need to repair damaged DNA in our genes to prevent the development of cancer and other diseases. Our cells therefore activate and send “repair-proteins”...

Im Focus: Dresdner scientists print tomorrow’s world

The Fraunhofer IWS Dresden and Technische Universität Dresden inaugurated their jointly operated Center for Additive Manufacturing Dresden (AMCD) with a festive ceremony on February 7, 2017. Scientists from various disciplines perform research on materials, additive manufacturing processes and innovative technologies, which build up components in a layer by layer process. This technology opens up new horizons for component design and combinations of functions. For example during fabrication, electrical conductors and sensors are already able to be additively manufactured into components. They provide information about stress conditions of a product during operation.

The 3D-printing technology, or additive manufacturing as it is often called, has long made the step out of scientific research laboratories into industrial...

All Focus news of the innovation-report >>>

Anzeige

Anzeige

Event News

Booth and panel discussion – The Lindau Nobel Laureate Meetings at the AAAS 2017 Annual Meeting

13.02.2017 | Event News

Complex Loading versus Hidden Reserves

10.02.2017 | Event News

International Conference on Crystal Growth in Freiburg

09.02.2017 | Event News

 
Latest News

A better way to measure the stiffness of cancer cells

01.03.2017 | Health and Medicine

Exploring the mysteries of supercooled water

01.03.2017 | Physics and Astronomy

Research team of the HAW Hamburg reanimated ancestral microbe from the depth of the earth

01.03.2017 | Life Sciences

VideoLinks
B2B-VideoLinks
More VideoLinks >>>