Gene Expression: _{Enzymes and hereditary conditions}

Shedding light on a hereditary disease: Waren Tay and Bernard Sachs

Although genes were still completely abstract in the late 18th and early 19th centuries, researchers were starting to recognize that certain diseases ran in families. One particularly devastating condition manifested itself with a range of symptoms in the central nervous system. Afflicted infants looked normal at birth, but gradually developed mental and physical retardation, leading to paralysis, blindness, deafness, and ultimately death, usually by age three. The disease has been around for ages, but only in the 19^th century had medicine advanced enough to recognize it. Various technological advances by 19^th century lens grinders allowed for major improvements in telescopes and microscopes, leading to some well-known discoveries in astronomy and biology. Alongside those improving telescopes and microscopes came a new invention: the ophthalmoscope. That’s the instrument that doctors use to examine the retinas of your eyes. It was invented in 1851, and by the 1880s was already the most important tool for ophthalmologists. Using one to examine a child with mental and physical retardation whose vision was also deteriorating, Waren Tay, a British ophthalmologist in London, noticed something in the retina that was not supposed to be there. He called it a “cherry red spot” (Figure 2), and in his report for a medical journal he noted that the child was Jewish.

An ocean away from Tay’s London practice, a New York pediatric neurologist, Bernard Sachs, was being sent all of the unusual neurologic cases in the city. Many of the patients were part of a new wave of immigrants to the city that included massive numbers of Jews from Central and Eastern Europe. After seeing a few cases of deteriorating physical and mental retardation, Sachs began looking at the brains of children who had died. Observing the same kind of swelling in the nerve cells from autopsy samples, Sachs came to realize that the patients were afflicted by the same disease. By questioning the parents of the children to see if they recalled stories of similar cases in their villages back in the old country, he also figured out that the condition ran in families of Jews. Calling the condition infantile amaurotic familial idiocy, Sachs noted that it skipped generations, usually more than one generation at a time, before showing up in another infant, such as the child or grandchild.

Gene expression

Eventually, Tay and Sachs (Figure 3) realized that they were studying the same condition. Today, it’s called Tay-Sachs disease, and the cherry red spot that Tay saw in his ophthalmoscope is a telltale sign. Reporting that the disease skipped generations, Sachs actually was implying that it displayed what Mendel termed a “recessive factor.” Just like the recessive shapes and colors of Mendel’s peapods, and just like blue eye color or straight hair, Tay-Sachs disease is caused by a specific version of a gene, but only if two copies of the version are present. While Sachs did not express his observations in Mendel’s terminology, this was around the time when Mendel’s laws were being rediscovered. Along with new instruments and methods shaping early 20th century science, those rediscovered laws beckoned to a new generation of geneticists.

But Mendel’s laws do not explain how dominance and recessivity work. How could it be that an infant gets a terrible disease, dies in childhood, and therefore does not grow up to have children, yet later the same disease reappears in a nephew or niece, or in a grandchild of the infant’s sibling? What is the path from a particular gene to the manifestation of a certain disease, condition, or trait? This process would not be understood until later researchers continued investigating the mechanism of gene expression over another several decades.

Enzymes and hereditary conditions: Archibald Garrod

Not far from Tay’s ophthalmology clinic in London, another medical doctor was conducting research that would lead to a major breakthrough in our understanding of gene expression. Archibald Garrod was studying people with a handful of medical conditions. Much more benign than Tay-Sachs disease, the conditions that piqued Garrod’s interest did not kill the patients as toddlers. At the same time, each condition came with a telltale trait. One condition that Garrod studied, called albinism, leaves children with no pigment in their hair, eyes, or skin. He was also fascinated by cystinuria, a condition characterized by frequent urinary stones beginning in early adulthood, and another condition that produces urine that darkens when left standing. Known as alkaptonuria, it typically is discovered after a parent notices dark stains in an infant’s diapers. Realizing that urine provided an easy way to study the chemistry of the body, Garrod also took urine samples from people whose health seemed perfectly normal. In doing so, he discovered another condition, called pentosuria, whose only sign is the presence of a certain kind of sugar in the urine.

Some of Garrod’s conditions also produce other effects that were not so easily recognized in those days. People with alkaptonuria, for instance, often develop trouble in large joints, disks of the spine, and heart valves as they age. But most of these problems appear long after the patients can grow up and have children of their own, and, in the case of pentosuria, there are no known detrimental effects on health. These features made investigating family connections much easier for Garrod than for Sachs.

Knowledgeable of the newly rediscovered Mendelian laws, Garrod hypothesized that a single recessive gene was the cause of each condition, and the gene was passed down in particular family lines (Figure 4). For instance, though pentosuria was the most benign disease of all the four that Garrod studied, it had something in common with the deadly Tay-Sachs disease; namely, it ran in Jewish families. Going beyond Mendel and Sachs, however, Garrod also suggested that for each condition, a recessive gene caused a deficiency of an enzyme whose normal role was to create, break down, or modify a particular chemical.

The role of enzymes and inborn errors of metabolism

It was an amazing stroke of insight, for Garrod was correct. Manufactured in all plant and animal cells, enzymes are catalysts that enable biochemical reactions to move forward at a faster rate, speeding up reactions that would take much longer. Enzymes are vital to many of the body’s critical functions; without them, organisms would not be able to survive and function. These predominantly protein-based molecules perform very specific tasks within the body (Figure 5). Understanding their role was key to a new understanding gene expression.

Each of the four conditions that Garrod studied really does result from a problem with a single enzyme. The same enzyme problem that causes dark urine in alkaptonuria also affects cartilage and other connective tissues throughout the body, thereby affecting the joints, spinal discs, and heart valves. This happens because the one enzyme that’s affected in alkaptonuria happens to control the breakdown of two of the 20 amino acids that life-forms use for just about everything. Garrod didn’t work out this amino acid chemistry, but studying the families of patients with alkaptonuria and the other abnormalities, he developed a concept called inborn errors of metabolism. The hypothesis was way ahead of its time, yet Garrod had no way to test it, so it did not catch on during his lifetime. Unlike da Vinci’s helicopter, though, Garrod’s vindication lay not four centuries into the future, but a mere four decades.

Making mutants: George Beadle and Edward Tatum

During the late 1930s, the final years of Garrod’s life, George Beadle was a young geneticist at Columbia University, where he was doing research on fruit flies called Drosophila melanogaster. Fruit flies, like humans, have noticeable differences in eye color that follow Mendelian inheritance. Using radiation to damage the Drosophila genes – whatever they were, for nobody yet knew their physical basis – Beadle was able to show that genes were related to eye color through a series of chemical reactions. Still, he couldn’t be sure whether the idea could apply to a wide range of traits and to life in general, or merely to eye color in fruit flies.

Teaming up with biochemist Edward Tatum in 1940, Beadle set aside the fruit flies in favor of Neurospora crassa, a type of bread mold (Figure 6). Like peas and people, fruit flies have two sets of chromosomes that carry genes for different characteristics. Thus, two genes encode the information for each trait, which is the cause of dominance and recessivity. Unlike fruit flies, N. crassa can produce little reproductive structures called spores that carry just one set of chromosomes, so dominance and recessivity do not come into play. Also, N. crassa offered another advantage. Studying fruit fly genetics, Beadle had to look at physical effects, like eye color, and picking up flies with tweezers can be time-consuming. N. crassa spores, on the other hand, could be placed on top of a nutrient-filled gel that has solidified, and Beadle could simply observe whether or not spores grew on the gel.

Since the answer was either “growing” or “not growing,” he could have hundreds of gel-filled plates, each with a spore. The nutrient gel contained only the minimal number of nutrients, the essential compounds, which the spores normally needed to grow (sugar, certain salts, and a vitamin called biotin). All other important chemical compounds the spores could make themselves, using the essential nutrients supplied in the gel as starting compounds. By using different nutrient mixtures, Beadle could observe whether a particular spore needed an extra nutrient, an ingredient not usually included in the gel since normal spores can make it themselves. Any spores needing an extra nutrient in order to grow could be considered abnormal. In genetics, these spores are called mutants, while the others (those able to grow with no extra ingredients) are called the wild type. If Garrod was right and each gene produced a certain enzyme, then damaging the gene for the enzyme that an organism used to make nutrient X would create a mutant organism that could grow only if nutrient X was supplied from the outside.

Although Beadle and Tatum did not know the physical basis of the genes, they were certain that each organism carried a whole lot of genes, probably thousands. In that case, how could they hope to create a mutant that depended on one and only one particular nutrient simply by zapping the organism with radiation? Actually, they weren’t sure that they could, but they understood the power in numbers. Like hoping to draw the queen of hearts from a shuffled deck of cards, you might get lucky if you try enough times. Likewise, when you can spread mold spores on a series of culture plates, you have more chances than when you’re picking up flies with tweezers. Even so, the scientists thought it could be a long shot, and so they made a deal. They would irradiate sample after sample and check mutant after mutant to see how they could grow or not grow with the addition or absence of particular nutrients. But they would set a limit of 5,000 attempts. If they got to that point without creating the mutant they needed, they would give up.

But they never had to give up because after just a few hundred attempts, they found a mutant that needed just one ingredient added to the usual growth mixture. That needed extra nutrient was arginine, one of the 20 amino acids that life-forms use as building blocks to make proteins. Normal N. crassa can make its own arginine, but Beadle and Tatum were able to create four different molds that could only survive when given arginine in their food. Using these strains, they were able to trace the chemical pathways connected with the mutated genes of the strains, ultimately demonstrating that each enzyme was made by one particular gene. Published in 1941, it was a milestone discovery that eventually would earn Beadle and Tatum the Nobel Prize. Their discovery was not limited to bread molds, for gradually it became clear that Tay-Sachs, all four of the conditions that Garrod studied, and a host of other familial disease were due to recessive gene mutants.

Gene mutants and health conditions

How does it work? Usually, for converting and breaking down chemicals in the body, enzymes are in fairly good supply. Like humans and peas, having two genes for everything, including for enzymes, means that you have a backup. If one gene of a pair is mutated and produces a defective enzyme or no enzyme at all, the individual still has the other gene, which makes enough enzyme to break down the chemical, convert the chemical to something else, or do whatever the enzyme does. Only an individual with two genes for the defective enzyme of alkaptonuria actually has the disease, just as two genes for a defective pigment are needed for a person to be an albino. A similar thing happens with human eye color. The gene for brown irises (the colored part of the eye) produces a dark pigment, which, if absent, leaves the iris blue. The dark color shows up, eliminating the blue, even if the individual has only one gene for brown eyes, which is why brown eyes are dominant. Blue eyes are recessive, because having them means you have no brown pigment at all, which only happens if both of your pigment genes are defective (Figure 7).

The principle also carries over to Tay-Sachs disease. Today, we know the disease is caused by an inability to break down a category of chemicals called lipids, specifically a special type of lipid called a GM2 ganglioside. Extremely important in membranes of nerve cells or neurons, GM2 ganglioside is broken down by an enzyme called HEXA. GM2 ganglioside gradually accumulates in the neurons of a child who makes no HEXA, but the accumulation takes time, which is why newborns with Tay-Sachs appear normal. Over months, however, the accumulating GM2 ganglioside causes the neurons to swell. Since the retina of the eye is made of the same kind of neurons that are in the brain, the retina swells in a particular pattern, and that’s what causes the cherry red spot that’s characteristic of Tay-Sachs disease, and eventually blindness. Similar swelling throughout the brain causes all of the other symptoms, and finally death.

From genes to protein enzymes

Beadle and Tatum’s demonstration that a defective gene leads to a defective enzyme proved that enzymes were made as a consequence of genes. Published in 1941, this was a watershed discovery in genetics that set the stage for other researchers to hone in on the physical basis of genes and on gene expression, the chemical pathways from genes to protein enzymes. The particular arrangement of atoms within a gene allows for storage of information. When that stored genetic information is used to make enzymes, the gene is expressed, and Beadle and Tatum set the stage for new researchers to discover how the gene expression process worked. The stories of those other researchers are recounted in the modules that focus on the genes and on each phase of the process leading to the manufacture of their products.

The gene products do not include only enzymes, but enzymes were the first gene products to be understood. All of the enzymes affected in genetic diseases, like those studied by Garrod, Tay, and Sachs, are proteins. Immensely versatile and complex, proteins come from amino acids, which are linked in a chain called a polypeptide. When properly folded, one or more polypeptides form a protein. In addition to being enzymes, proteins take on a variety of roles, from providing structure for biological tissues to carrying important molecules around the body and in and out of cells.

The Central Dogma of molecular biology

Using the Beadle-Tatum discovery as a starting point, biologists during the 1940s and 50s figured out not just that DNA carried the genes, but they started to get an idea of how those genes were replicated and passed from generation to generation. Soon after that they learned that amino acids were put together into polypeptides using a set of rules called the genetic code, which is nearly the same for all life-forms on Earth. They also learned that the genetic code was not a way for cells to translate genetic information in DNA directly into chains of amino acids to make proteins. Instead, there are molecules called RNA that must be made as intermediaries along the way from DNA to the polypeptides that fold into proteins. The process of using DNA to make RNA, and then RNA to make polypeptides is one-directional. Never is a sequence of amino acids of a polypeptide used as a message for making either RNA or DNA, and only in certain viral infections is RNA ever used to make DNA. Known as the Central Dogma of molecular biology, this one-way process is universal to all organisms.

The one-directional nature of the movement of genetic information, which scientists came to understand from the 1950s-1970s, rests upon Beadle and Tatum’s 1941 watershed discovery. Before anyone could identify DNA as the physical basis of genes, and before anyone could reveal the chemistry carrying the genetic messages from DNA to RNA, and finally to the amino acids that make a polypeptide, somebody had to show what chemical product genes were actually affecting. And in the case of the various inherited diseases, it was enzymes. This supported the hypothesis of “one gene, one enzyme,” which was expanded to “one gene, one polypeptide” after it was realized that non-enzyme proteins were also made using genes, and eventually it had to be expanded again. Although many RNA molecules carry actual genetic messages from DNA that are used to make polypeptides, the job of some other RNA molecules is to help with the process. Since the helper RNA molecules also are made from genes, it’s not accurate to say that all genes code for some kind of protein product. Thus, today we say "one gene, one RNA."

Even before Beadle and Tatum could prove “one gene, one enzyme” with their painstaking bread mold experiments, somebody had to imagine a connection between genes and enzymes in the first place. Poor Archibald Garrod had died in 1936, just five years short of the Beadle and Tatum publication that vindicated the idea of inborn errors of metabolism. But Beadle and Tatum did remember Garrod. Inspired by their predecessor, Beadle named him at their 1958 Nobel Prize acceptance as the ultimate inspiration for their work.