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FEATURE

PITT RESEARCHER PAULA GRABOWSKI
EXPLORES THE GENETIC BOND THAT WE ALL SHARE.


THE SPLICE OF LIFE
WRITTEN BY CURT WOHLEBER
YOUR MEMORY PROBABLY doesn't go back that far, but you started out life as a single cell, a fertilized ovum resting in the womb. Before long, however, you divided in two. Then those two cells divided in turn, and so on. So , for a time, you were a cluster of multiplying cells, each one pretty much the same as the other.

But by the time you were born, your cells -- now numbering in the trillions -- had astonishing diversity: blood cells, skin cells, brain cells -- an incredibly vast and varied catalog of cells necessary to maintain an organism as complex and versatile as you.

And this diversification does not end at birth. Minute by minute, hour by hour, new generations of cells continually take on new identities and undertake new functions to meet the body's changing needs. Identical cells in the bone marrow multiply into all the different kinds of blood cells used by the circulatory system to transport oxygen, fight infections, and dispose of waste chemicals. Cells in the brain will retool to manufacture various kinds of chemical messengers to perform the functions needed fo r thinking, perceiving, and remembering.

"What makes a cell reach its destiny?" asks Paula Grabowski, Pitt professor of biological sciences, sitting in her cheerful hut orderly Langley Hall office. Grabowski is one of the world's leading researchers in pursuit of this question. Her work got a bi g boost last year when she became one of 48 scientists named a 1994 Howard Hughes Medical Institute Investigator. This significant honor confers not only high prestige but also several million dollars of research funding over a five-year period. Grabowski hopes that this substantial award from the Hughes Medical Institute, the nation's largest private philanthropic organization, will bring her closer to understanding the remarkable genetic process of cell differentiation. In the lab adjoining her office, her team of grad students and research assistants are helping her find answers to her questions. They spin solutions in centrifuges to tease apart reaction products and sort molecules according to size by placing chemicals in an electrically conductive g el. It doesn't look too dramatic: no lightning bolts and crackling Van de Graaff generators readying to jolt inanimate matter to life. No 50-mile-long accelerators hurtling subatomic particles to near-warp speed. But research at this lab and others like i t is slowly making sense of basic life processes that for earlier generations of scientists could only be described with shrugs and words like ''miracle.''

If your cells could talk, you'd probably wish they'd shut up. The activity within a cell can be likened to the scramble of a Wall Street trading pit. The cell constantly manufactures useful substances while tearing apart surplus chemicals and waste produc ts and disposing of or recycling the pieces. And it all happens blindingly fast, many times over even as you are sleeping or watching America's Most Wanted or hunting the car keys. The biological machinery of an entire cell -- the nucleus, cytoplas m, mitochondria, vacuoles, and the rest of those terms you memorized cramming for that high school biology test -- can be duplicated during cell division in a matter of seconds.

Orchestrating the cell's operations are an immense series of coded instructions inscribed in its nucleus. The cell continually reads and interprets these instructions to carry out its work. These instructions are called genes. Genes are why people who are related may resemble each other, or at least have the same color eyes or hair. Genes carry information from one generation to the next: blue eyes, brown hair, and put the heart right here, under the ribcage.

But how is it, exactly, that a gene is able to carry out the enterprise of making the living, breathing, thinking organisms we are? How is it that a gene can direct the mystery that is life?

TO BE TECHNICAL ABOUT it, genes are made of deoxyribonucleic acid, or DNA. A DNA molecule can be visualized as a long ladder twisted into a spiral. Each "rung" of this ladder is a letter in the language of the genes. There are four letters in the genetic alphabet of DNA, each representing a different kind of rung.

Your genetic blueprint contains some 200 million rungs. Transcribed as text, this information would fill up roughly 30,000 pages of this magazine. And all that information is encoded in every one of the trillions of cells in your body. As a medium for sto ring information, DNA makes CD-ROMs look primitive.

A gene is a sequence of rungs along the DNA ladder. If each rung is a letter, then three rungs make up a word. A gene can be thousands of "words" long -- an intricately complicated sentence, if you will. On top of that, it's a sentence that is intersperse d with nonsense. On the other hand, the question that drives Grabowski's work is simple and elegant: How does nature put the sentence back together? How does nature make sense of genes?

But first a word about proteins. The sentence made by the gene contains a message to the cell, telling it how to put together a protein. So what are proteins? Well, they're nothing less than the chemicals that make life happen.

Some proteins carry oxygen through the bloodstream. Others attack foreign organisms. Still others serve as key components of hair, muscle, skin, and bone. The vast majority of proteins, however, are enzymes. These are large molecules that speed up chemical reactions, either helping atoms and molecules bind together to form needed substances, or helping molecules break down into easily manag ed pieces.

The chemical reactions driven by protein enzymes grow bodily tissues, digest food and turn it into energy, fight diseases and heal wounds, recycle used chemicals in the body, and process the oxygen we breathe. Proteins are even involved in the chemical re actions believed to govern thought and emotion.

The genetic blueprint, the map of all the proteins used by the body, exists in every cell of the body. Think about it: within cells at the tip of your forefinger, for instance, reside secrets of your pancreas, your nervous system, your gastrointestinal tr act.

But how do individual cells know how to make the right protein at the right time? Every cell does not need every protein all the time. A skin cell does not need proteins used by the liver.

How the cell properly coordinates the manufacture of proteins is called gene regulation. Some types of gene regulation are quite simple: Many genes that code for unneeded proteins are simply "turned off."

Grabowski's lab studies a more complex type of gene regulation called RNA splicing. RNA splicing is sort of a Total Quality Management approach to gene regulation: The final decisions about what kinds of proteins should be assembled aren't made in the woo dpaneled offices of the executive chromosomes, but out on the noisy shop floor of the nucleus, where copies of genes are interpreted and edited to meet the needs of the moment.

Why study RNA splicing? Characteristically, Grabowski pauses a moment to consider the question carefully, then she notes that discoveries about gene regulation could provide valuable insight into the detection or treatment of certain diseases. Then she co mes clean: "I'm doing this mainly because it's a lot of fun to ask questions."

WHICH CAME FIRST, THE chicken or the egg? The biological silences have a variation: which came first, DNA or protein? You see, among the many tasks performed by proteins is assembling DNA molecules. But DNA contains the in formation needed to make proteins. So which came first?

Scientists pondering the origin of life have speculated that proteins punched in first, accidentally coming into being billions of years ago in a lucky collision of amino acid molecules. But when Grabowski was a doctoral student at the University of Color ado at Boulder, she participated in an unexpected discovery that stunned chemists worldwide and suggested a very different answer about the origin of life.

This discovery involves RNA, which is a chemical cousin to DNA. To make a protein designated by a specific gene, the cell makes a copy of the gene in the form of an RNA molecule. The RNA copy goes to one of the cell's ribosomes, which are its protein-maki ng factories. The ribosome "reads" the rungs of RNA. Every three-letter RNA "word" stands for a particular piece of the protein. Reading the RNA copy allows the ribosome to put together the pieces of the protein in correct order.

Until the 1980s, scientists thought that all RNA ever did was serve as a sort of carbon copy of DNA.

However, Grabowski and another postdoctoral researcher in the lab of Nobel-prize-winning scientist Thomas Cech were hunting down proteins when they performed independent experiments that revealed, quite by accident, that RNA molecules could do more than f unction as passive carriers of information: They could function as enzymes, carrying out chemical reactions.

"It was a heretical notion," recalls Cech, who was Grabowski's dissertation advisor at the time. Scientists had thought only proteins could function as enzymes. No one would have guessed that RNA had such versatility. The discovery upset decades of conven tional wisdom and forced scientists to reconsider certain long-accepted notions about some basic cellular processes.

"It was a very bizarre finding," says Grabowski. "Nothing like that had been reported before, and the first thing we thought was that the observation was wrong."

Cech, Grabowski, and the other researchers originally figured that undetected proteins had probably carried out the reactions they observed in the test tube. But they performed additional experiments, testing various alternative hypotheses and analyzing t he reaction step by step until they had firm evidence that the original finding was correct.

As a result of this discovery, many scientists now suspect that the very first microscopic life forms on Earth were RNA-based, with RNA molecules performing double duty as both carriers of genetic information and as the organism's chemical workhorses, pro moting the vital chemical reactions needed to maintain life.

This new understanding of RNA enzymes, to which Grabowski contributed, earned Cech the 1989 Nobel prize in chemistry. In 1993, another of Grabowski's mentors, MIT biologist Philip Sharp, was awarded the Nobel prize in physiology.

"I can choose people who win the Nobel Prize," quips Grabowski. "This is the one talent I seem to have."

"Actually," she goes on, offering a revealing statement, "I picked Cech and Sharp to work with because of the way they think, more than the actual research projects they were doing."

Grabowski studied chemistry in college, drawn by the analytical thinking required by the discipline. As a graduate student at Boulder, she attended informal presentations given by faculty members who would talk about their research. "I listened more for h ow they were thinking about the problem than for what they were actually doing," she recalls. "That's why I wanted to work with Tom Cech. He takes an important problem, thinks very cleanly about it, and is able to design simple experiments that give clear -cut answers. That's exactly what you want to do in science, and for graduate students, that's the thing to learn--how to design experiments that distinguish possibilities. "

Grabowski was similarly impressed by Philip Sharp's thinking and went to work for him at MIT after receiving her doctorate in 1983. In the late 1970s, Sharp had made a discovery that propelled the study of gene regulation -- Grabowski's specialty -- in an entirely new direction.

Sharp and his team of researchers had discovered that individual genes are interrupted by stretches of "junk DNA," chemical gibberish that does not contain information needed to make proteins. These sections of "junk" are called introns. After a gene is c opied into RNA, these introns have to be snipped out. Otherwise, the cell's protein-making factories, the ribosomes, would misread the RNA and assemble faulty proteins.

Working at MIT under Sharp, Grabowski helped identify the bodies that edit the introns out of RNA molecules then splice the loose ends together. Dubbed spliceosomes, these bodies are themselves composed of RNA bundled up with proteins.

All well and good, but why is there all this junk in our genes to begin with?

Apparently, introns serve as markers, dividing a gene into individual sections. When editing the RNA copy of the gene, spliceosomes can piece together these sections in different combinations. When read by the ribosomes, these different combinations yield different kinds of proteins.

Previously, scientists had believed that each gene coded for a single protein. But through RNA splicing, the cell can use a single gene to create different kinds of proteins -- composing, as it were, variations on a theme. Grabowski's current research foc uses on how RNA is spliced in different ways.

RNA splicing may play a significant role in evolution by providing cells with a quick way to adapt to changing conditions. Scientists as yet have only a rudimentary understanding of how a cell controls RNA splicing. Grabowski's lab is working to uncover m olecules that govern RNA splicing in neurons, which are the cells that do most of the important work in the brain. Neurons provide fertile ground for the study of RNA splicing because they are highly specialized -- after all, a lot of different things go on even in simple brains -- and they need to produce many different kinds of chemical messengers to coordinate such things as the perception of pain or the formation of ideas.

The research of Grabowski and other scientists in her field will almost certainly yield medical advances and other practical benefits, but perhaps just as importantly, it will bring us to a keener understanding and appreciation of how life works. The book of our genes contains what may be the most compelling story ever told, the epic of life, the unfolding processes of growth, change, and death. Our cells continually read this story, but now we are beginning to have the opportunity to listen to the story ourselves.


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