Catch Up August 18, 2000

I just started reading a book by Freeman Dyson (The Sun, The Genome and The Internet: Tools of Scientific Revolutions) detailing his views on the major scientific and technological work that will be done in this century. He recognizes the fact that he will be wrong but he uses this device to expand on some of his ideas regarding how science is done. I am only 30 pages into it, and I am sure I'll discuss it in greater detail when I have finished it, but he already has an interesting point of view.

He believes that science generally advances because of new tools that become available, rather than by simply the testing of new theories. Because the new tools allow us to ask, and answer questions, that were unthinkable previously, and thus lead to new and novel information. Old theories must be overthrown, not simply because new theories arise, but because new data is generated that the old theories can not explain.

This week I'm going to revisit some of the topics I have mentioned in previous columns, hoping to update you with some new data. As we all know, science continues to march on. and I find it intriguing to see what new information may have been generated, and what its effect may be on earlier data.

Last week, I discussed an approach using NMR (MRI) technology to examine gene expression in living animals. EgadMe was the contrast agent used to visualize the cells. An alert reader wrote me about another approach which also uses expression of a transgene for visualization. The main problem with EgadMe is getting it inside the cell. The work I described previously got around this by simultaneously co-injecting the mRNA for a gene coding for the enzyme of choice and the EgadMe. This may not be useful as a general technique. The present paper, published in the March 2000 Nature Medicine, uses a novel approach to get the contrast agent inside cells – simply injecting the contrast agent into the blood stream and letting the appropriate cells take up the agent.

They use an engineered transferrin receptor (ETR) gene to internalize 3 nm monocrystalline iron oxide nanoparticles (MION) that are fed intravenously into a mouse. Cells containing MION have an altered signal using MRI than those without MION. Mice were injected with gliosarcoma cells that did or did not express ETR. After 12 days, tumors were easily seen. The mice were then given MION and it is very easy to discern the ETR+ cells from ETR-.

Finally, it was also possible to correlate ETR gene expression with the MRI signal intensity. The more ETR expressed, the greater the effect on the MRI signal. So, it is possible that different promoter elements can be used to drive ETR expression, allowing the direct detection of tissue expression via MRI. The ability to monitor the expression of exogenous genes used in gene therapy holds a lot of promise. And it appears possible that this approach could detect single cells. Pretty cool.

Carbon isotope levels continue to be used to examine extinction episodes in the Earth's history. A recent paper looks at such an event during the Jurassic, about 150 million years ago. There is a very large 'negative excursion' in the ¹³C ratios at this time. This work seems to demonstrate that a large amount of 'light' carbon entered the carbon cycle during this time period. What is interesting in this work is the cause of this accumulation of ¹²C.

For many years there was a mystery surrounding the carbon cycle. Mass action models indicated that there must be a huge reservoir of carbon in the ocean, but the amount of dissolved CO₂ in the ocean could not account for it. It turns out that the carbon is not sequestered as CO₂ but as methane. Large amounts of methane are found in gas hydrates, in which solid crystals of water and methane form stable structures at a variety of temperatures and pressures. These hydrates are enriched for ¹²C. Release of this methane, due to deep-water warming, would have a devastating effect on the carbon cycle and on greenhouse warming. This is because the methane would be oxidized to CO₂ that would increase the greenhouse effect.

However, plants would also take up this CO₂. So, Hesselbo et al.also look at the carbon-isotope ratios in various organic deposits from the same time period. They show that the expected pulse of light carbon is found here too. They also show that the most likely site of such an influx comes from methane hydrates rather than volcanoes. The implications for today's climate, where global warming has the potential for similar release of methane, is obvious. So, keep an eye on research looking at these methane hydrates and deep-sea warming.

A final series of papers in the August 11 issue of Science clarify the role of the ribosome in protein synthesis. Some have proposed that the ribosome is a relic of the RNA world. The only enzymatic activity of the ribosome, the joining of the 2 amino acids, is not present in any ribosomal protein. The first paper describes the use of X-ray crystallography to determine the complete structure of the large subunit of the ribosome to 2.4 Å resolution. 27 of the 31 ribosomal proteins could be seen. The rRNA structure is very extended and most of the proteins follow these extensions, looking for all the world like simple structural scaffolds for the rRNA. The only area that appears to be devoid of protein is the active site of peptide bond formation.

A second paper attempts to identify the active site by soaking the crystals used for structure determination with substrate analogs. These analogs found their way to the active site and subsequent X-ray diffraction identified this region. While the location of this active site was expected, the nearest protein is over 18 Å away. The proposal that the catalytic activity of the ribosome rests purely in the rRNA sequences, that the ribosome is a ribozyme, fits the new data extremely well. In fact, a third paper identifies a single invariant adenosine residue as the critical site for the catalysis.

A proposed mechanism for a general acid-base catalysis is given, for all you electron-pushers. This is similar to the mechanism of many proteases, with the adenosine filling the role of a histadine. One feature of most acid-base catalysts is a pKa close to neutral. However, the appropriate nitrogen of adenosine normally has a pKa of 3.5. But, knowing which adenosine to look at now, the authors were able to show that the pKa of the critical adenosine, A2451, is perturbed by its immediate microenvironment and has a pKa of about 7.6. Almost exactly the expected pKa.

A lot of future work will be done investigating the exact mechanism of this reaction, but the idea that a fundamental aspect of protein translation is actually driven by a ribozyme has to be one of the most expected 'unexpected' results of the last 25 years. Recent investigations indicated that this was a real possibility, but this work makes it definite.

RNA molecules have gone from being passive carriers to THE most important moiety in biology. We would most likely not be here if RNA catalysts had not evolved. Everything else in biology is just 'small' evolutionary changes of this revolutionary process. (Just a small overstatement, huh?)

These 3 topics are linked by the idea that technology will drive new explorations in biology. Although biological investigations using X-ray crystallography has been around for almost 50 years, modern technology now allows us to visualize enormously complex structures. It has now verified something that other data had only suggested. ¹³C levels are telling us a lot about the climate of ancient Earth, as well as its effect on life. The ability to track gene expression in living organisms will definitely open new vistas in our understanding.

Modern biology is driven by technology. Data rules. And we need much more technology today to get the data than we did 100 years ago, or even 25 years ago or even 2 years ago. I'm not sure what Freeman Dyson will discuss in his book, but I know that the coming years will require biologists who are extremely comfortable with high technology. They will be able to visualize the inner workings of machines as well as cells. They may even LIKE math. For the rest of us, we can only hope that we continue to play catch up or we will be forced into our rocking chairs several decades too soon.