Occasionally, unusual factors influence whether or not a child will be born with a genetic disease. An example is the molecular error that causes Fragile X syndrome, a rare condition associated with mental retar dation. The mutation leading to a fragile X chromosome is not a typical DNA typ ing mistake, in which nucleotides are switched around or dropped, or one of them is switched for another nucleotide. Instead, it is a kind of stutter by the DNA polymerase enzyme that copies DNA. This stutter creates a string of repeats of a DNA sequence that is composed of just three nucleotides, CGG. Some people have only one repeat of the CGG nucleotide triplet. Thus, they have two copies of the repeat in a gene, and the extra sequence reads CGGCGG. Others have more than a thousand copies of the repeat. These people are the most severely affected. The number of triplet repeats seems to increase as the chromosome is passed down through several genera tions. Thus, the grandsons of a man with a fragile X chromosome, who is not himself affected, have a 40 percent risk of retardation if they inherit the repeatcontaining chromosome. The risk for greatgrandsons is even higher: 50 percent. Intrigued by the evidence that triplet repeats can cause genetic disease, scien tists have searched for other examples of disorders associated with the DNA expansions. To date, more than a dozen such disorders have been found, and all of them affect the nervous system. Analysis of the rare families in which such diseases are common has revealed that expansion of the triplet repeats is linked to something called genetic anticipation, when a disease’s symptoms appear earlier and more severely in each successive generation.
Battle of the SexesA process called imprinting, which occurs natu rally in our cells, provides another example of how epigenetics affects gene activity. With most genes, the two copies work exactly the same way. For some mammalian genes, how ever, only the mother’s or the father’s copy is switched on regardless of the child’s gender. This is because the genes are chemically marked, or imprinted, during the process that generates eggs and sperm. As a result, the embryo that emerges from the joining of egg and sperm can tell whether a gene copy came from Mom or Dad, so it knows which copy of the gene to shut off. One example of an imprinted gene is insulin like growth factor 2 (Igf2), a gene that helps a mammalian fetus grow. In this case, only the father’s copy of Igf2 is expressed, and the mother’s copy remains silent (is not expressed) throughout the life of the offspring. Scientists have discovered that this selective silencing of Igf2 and many other imprinted genes occurs in all placental mammals (all except the platypus, echidna and marsupials) examined so far, but not in birds. Why would nature tolerate a process that puts an organism at risk because only one of two copies of a gene is working? The likely reason, many researchers believe, is that mothers and fathers have competing interests, and the battle field is DNA! The scenario goes like this: It is in a father’s interest for his embryos to get bigger faster, because that will improve his offspring’s chances of survival after birth. The better an individual’s chance of surviving infancy, the better its chance of becoming an adult, mating and passing its genes on to the next generation. Of course mothers want strong babies, but unlike fathers, mothers provide physical resources to embryos during pregnancy. Over her lifetime, a female is likely to be pregnant several times, so she needs to divide her resources among a num ber of embryos in different pregnancies. Researchers have discovered over 200 imprinted genes in mammals since the first one was identified in 1991. We now know that imprinting controls some of the genes that have an important role in regulating embryonic and fetal growth and allocat ing maternal resources. Not surprisingly, mutations in these genes cause serious growth disorders. Marisa Bartolomei of the University of Pennsylvania School of Medicine in Philadelphia is trying to figure out how Igf2 and other genes become imprinted and stay silent throughout the life of an individual. She has already identified sequences within genes that are essential for imprinting. Bartolomei and other researchers have shown that these sequences, called insula tors, serve as “landing sites” for a protein that keeps the imprinted gene from being transcribed.
Starting at the EndWhen we think of DNA, we think of genes. However, some DNA sequences are different: They don’t encode RNAs or proteins. Introns, described in page 10, are in this category. Another example is telomeres—the ends of chromosomes. There are no genes in telomeres, but they serve an essential function. Like shoelaces without their tips, chromosomes with out telomeres unravel and fray. And without telomeres, chromosomes stick to each other and cause cells to undergo harmful changes like divid ing abnormally. Researchers know a good deal about telo meres, dating back to experiments performed in the 1970s by Elizabeth Blackburn, a basic researcher who was curious about some of the fundamental events that take place within cells. At the time, Blackburn, now at the University of California, San Francisco, was working with Joseph Gall at Yale University. For her experi mental system, she chose a singlecelled, ponddwelling organism named Tetrahymena. These tiny, pearshaped creatures are covered with hairlike cilia that they use to propel them selves through the water as they devour bacteria and fungi. Tetrahymena was a good organism for Blackburn’s experiments because it has a large number of chromosomes — which means it has a lot of telomeres! Her research was also perfectly timed, because methods for sequencing DNA were just being developed. Blackburn found that Tetrahymena’s telomeres had an unusual nucleotide sequence: TTGGGG, repeated about 50 times per telomere. Since then, scientists have discovered that the telomeres of almost all organisms have repeated sequences of DNA with lots of Ts and Gs. In human and mouse telomeres, for example, the repeated sequence is TTAGGG. The number of telomere repeats varies enor mously, not just from organism to organism but in different cells of the same organism and even within a single cell over time. Blackburn reasoned that the repeat number might vary if cells had an enzyme that added copies of the repeated sequence to the telomeres of some but not all chromosomes. With her thengraduate student Carol Greider, now at Johns Hopkins University, Blackburn hunted for the enzyme. The team found it and Greider named it telomerase. Blackburn, Greider and Jack Szostak of Harvard Medical School in Boston shared the 2009 Nobel Prize in physiology or medicine for their discov eries about telomeres and telomerase. As it turns out, the telomerase enzyme con sists of a protein and an RNA component, which the enzyme uses as a template for copying the repeated DNA sequence. What is the natural function of telomerase? As cells divide again and again, their telomeres get shorter. Most normal cells stop dividing when their telomeres wear down to a certain point, and eventually the cells die. Telomerase can counter act the shortening. By adding DNA to telomeres, telomerase rebuilds the telomere and resets the cell’s molecular clock. The discovery of telomerase triggered new ideas and literally thousands of new studies. Many researchers thought that the enzyme might play important roles in cancer and aging. Researchers were hoping to find ways to turn telomerase on so that cells would continue to divide (to grow extra cells for burn patients, for example), or off so that cells would stop dividing (to stop cancer, for instance). So far, they have been unsuccessful. Although it is clear that telomerase and cellular aging are related, researchers do not know whether telo merase plays a role in the normal cellular aging process or in diseases like cancer. Recently, however, Blackburn and a team of other scientists discovered that chronic stress and the perception that life is stressful affect telomere length and telomerase activity in the cells of healthy women. Blackburn and her coworkers are currently conducting a longterm, followup study to confirm these intriguing results.
The Other Human GenomeBefore you think everything’s been said about DNA, there’s one little thing we didn’t mention: Some of the DNA in every cell is quite different from the DNA that we’ve been talking about up to this point. This special DNA isn’t in chromo somes—it isn’t even inside the cell’s nucleus where all the chromosomes are! So where is this special DNA? It’s inside mito chondria, the organelles in our cells that produce the energyrich molecule adenosine triphosphate, or ATP. Mendel knew nothing of mitochondria, since they weren’t discovered until late in the 19th century. And it wasn’t until the 1960s that researchers discovered the mitochondrial genome, which is circular like the genomes of bacteria. In human cells, mitochondrial DNA makes up less than 1 percent of the total DNA in each of our cells. The mitochondrial genome is very small—containing only about three dozen genes. These encode a few of the proteins that are in the mitochondrion, plus a set of ribosomal RNAs used for synthesizing proteins for the organelle. Mitochondria need many more proteins though, and most of these are encoded by genes in the nucleus. Thus, the energyproducing capa bilities of human mitochondria—a vital part of any cell’s everyday health—depend on coordi nated teamwork among hundreds of genes in two cellular neighborhoods: the nucleus and the mitochondrion. Mitochondrial DNA gets transcribed and the RNA is translated by enzymes that are very different from those that perform this job for genes in our chromosomes. Mitochondrial enzymes look and act much more like those from bacteria, which is not surprising because mitochondria are thought to have descended from freeliving bacteria that were engulfed by another cell over a billion years ago. Scientists have linked mitochondrial DNA defects with a wide range of agerelated diseases including neurodegenerative disorders, some forms of heart disease, diabetes and various cancers. It is still unclear, though, whether dam aged mitochondria are a symptom or a cause of these health conditions. Scientists have studied mitochondrial DNA for another reason: to understand the history of the human race. Unlike our chromosomal DNA, which we inherit from both parents, we get all of our mitochondrial DNA from our mothers. Thus, it is possible to deduce who our mater nal ancestors were by tracking the inheritance of mutations in mitochondrial DNA. For reasons that are still not well understood, mutations accumulate in mitochondrial DNA more quickly than in chromosomal DNA. So, it’s possible to trace your maternal ancestry way back beyond any relatives you may know by name—all the way back to “African Eve,” the ancestor of us all!
In the early 1970s, scientists discovered that they could change an organism’s genetic traits by putting genetic material from another organ ism into its cells. This discovery, which caused quite a stir, paved the way for many extraordinary accomplishments in medical research that have occurred over the past 35 years. How do scientists move genes from one organism to another? The cutting and pasting gets done with chemical scissors: enzymes, to be specific. Take insulin, for example. Let’s say a sci entist wants to make large quantities of this protein to treat diabetes. She decides to transfer the human gene for insulin into a bacterium, Escherichia coli, or E. coli, which is commonly used for genetic research (see Living Laboratories, page 46). That’s because E. coli reproduces really fast, so after one bacterium gets the human insulin gene, it doesn’t take much time to grow millions of bacteria that contain the gene. The first step is to cut the insulin gene out of a copied, or “cloned,” version of the human DNA using a special bacterial enzyme from bacteria called a restriction endonuclease. (The normal role of these enzymes in bacteria is to chew up the DNA of viruses and other invaders.) Each restric tion enzyme recognizes and cuts at a different nucleotide sequence, so it’s possible to be very pre cise about DNA cutting by selecting one of several hundred of these enzymes that cuts at the desired sequence. Most restriction endo nucleases make slightly staggered incisions, resulting in “sticky ends,” out of which one strand protrudes.The next step in this example is to splice, or paste, the human insulin gene into a circle of bacterial DNA called a plasmid. Attaching the cut ends together is done with a different enzyme (obtained from a virus), called DNA ligase. The sticky ends join back together kind of like jigsaw puzzle pieces. The result: a cutandpasted mixture of human and bacterial DNA. The last step is putting the new, recombi nant DNA back into E. coli and letting the bacteria reproduce in a petri dish. Now, the scientist has a great tool: a version of E. coli that produces lots of human insulin that can be used for treating people with diabetes. So, what is cloning? Strictly speaking, it’s making many copies. However, the term is more commonly used to refer to making many copies of a gene, as in the E. coli example above. Researchers can also clone entire organisms, like Dolly the sheep, which contained the identical genetic material of another sheep.
The Tools of Genetics: Recombinant DNA and Cloning